U.S. patent application number 13/391144 was filed with the patent office on 2012-08-16 for methods of using cd44 fusion proteins to treat cancer.
This patent application is currently assigned to CENTRE HOSPITALIER UNIVERSITAIRE VAUDOIS. Invention is credited to Ivan Stamenkovic, Qin Yu.
Application Number | 20120207753 13/391144 |
Document ID | / |
Family ID | 43607294 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120207753 |
Kind Code |
A1 |
Yu; Qin ; et al. |
August 16, 2012 |
METHODS OF USING CD44 FUSION PROTEINS TO TREAT CANCER
Abstract
Pharmaceutical compositions and methods for treating cancer
using CD44 antagonists are disclosed. In certain aspects, these
pharmaceutical compositions and methods include treating a mammal
having a cancer, such as glioma, colon cancer, breast cancer,
prostate cancer, ovarian cancer, lung cancer, renal cell carcinoma,
gastric cancer, esophageal cancer, head cancer, neck cancer,
pancreatic cancer, or melanoma, with a CD44 fusion protein. These
CD44 fusion proteins include CD44-Fc fusions and can be used to
detect hyaluronan.
Inventors: |
Yu; Qin; (New York, NY)
; Stamenkovic; Ivan; (Lausanne, CH) |
Assignee: |
CENTRE HOSPITALIER UNIVERSITAIRE
VAUDOIS
Lausanne
NY
MOUNT SINAI SCHOOL OF MEDICINE OF NEW YORK
New York
|
Family ID: |
43607294 |
Appl. No.: |
13/391144 |
Filed: |
August 16, 2010 |
PCT Filed: |
August 16, 2010 |
PCT NO: |
PCT/US10/45635 |
371 Date: |
May 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61274813 |
Aug 21, 2009 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
600/1 |
Current CPC
Class: |
C12N 2310/11 20130101;
G01N 2800/56 20130101; C12N 15/1138 20130101; C12N 2310/531
20130101; G01N 33/57484 20130101; C07K 14/70585 20130101; C07K
2319/30 20130101; A61P 35/00 20180101; C12N 15/62 20130101; C12N
2310/14 20130101 |
Class at
Publication: |
424/134.1 ;
600/1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61M 37/00 20060101 A61M037/00; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The United States Government has certain rights to this
invention by virtue of funding received from the Department of
Defense, Army Medical Research, Grant No. W81XWH-06-1-0246 and
National Institute of Health, National Cancer Institute Research,
Grant No. R01CA135158-01A1.
Claims
1. A method for treating a cancer in a mammal, comprising
administering to the mammal in need of such treatment an effective
amount for treating the cancer a CD44 fusion protein comprising the
constant region of human IgG1 fused to an extracellular domain of
CD44, wherein the cancer is a member selected from the group
consisting of glioma, colon cancer, breast cancer, prostate cancer,
ovarian cancer, lung cancer, renal cell carcinoma, gastric cancer,
esophageal cancer, head-neck cancer, pancreatic cancer, liver
cancer, and melanoma.
2. (canceled)
3. (canceled)
4. The method according to claim 1, wherein the glioma is a
glioblastoma multiforme.
5. The method according to claim 1, wherein the mammal is a
human.
6. The method according to claim 1, wherein the extracellular
domain of CD44 is a member selected from the group consisting of
CD44s, CD44v3-v10, CD44v8-v10, CD44v4-v10, CD44v5-v10, CD44v6-v10,
CD44v7-v10, CD44v9-v10, CD44v10, CD44v9, CD44v8, CD44v7, CD44v6,
CD44v5, CD44v4, CD44v3, CD44sR41A, CD44v3-v10R41A, CD44v8-v10R41A,
CD44v4-v10R41A, CD44v5-v10R41A, CD44v6-v10R41A, CD44v7-v10R41A,
CD44v9-v10R41A, CD44v10R41A, CD44v9R41A, CD44v8R41A, CD44v7R41A,
CD44v6R41A, CD44v5R41A, CD44v4R41A, and CD44v3R41A.
7. The method according to claim 6, wherein the extracellular
domain of CD44 is CD44v3-v10.
8. The method according to claim 6, wherein the extracellular
domain of CD44 is CD44v8-v10.
9. (canceled)
10. A pharmaceutical composition comprising: a) a CD44 fusion
protein comprising the constant region of human IgG1 fused to an
extracellular domain of CD44, wherein the extracellular domain of
CD44 is a member selected from the group consisting of CD44v3-v10,
CD44v8-v10, CD44s, CD44v4-v10, CD44v5-v10, CD44v6-v10, CD44v7-v10,
CD44v9-v10, CD44v10, CD44v9, CD44v8, CD44v7, CD44v6, CD44v5,
CD44v4, CD44v3, CD44sR41A, CD44v3-v10R41A, CD44v8-v10R41A,
CD44v4-v10R41A, CD44v5-v10R41A, CD44v6-v10R41A, CD44v7-v10R41A,
CD44v9-v10R41A, CD44v10R41A, CD44v9R41A, CD44v8R41A, CD44v7R41A,
CD44v6R41A, CD44v5R41A, CD44v4R41A, and CD44v3R41A; and b) a
pharmaceutically acceptable carrier or diluent.
11. The pharmaceutical composition of claim 10, wherein the
extracellular domain of CD44 is CD44v3-v10.
12. The pharmaceutical composition of claim 10, wherein the
extracellular domain of CD44 is CD44v8-v10.
13. (canceled)
14. The pharmaceutical composition of claim 10, wherein the
extracellular domain of CD44 is CD44v3-v10R41A.
15. The pharmaceutical composition of claim 10, wherein the
extracellular domain of CD44 is CD44v8-v10R41A.
16. The pharmaceutical composition of claim 10, wherein the
extracellular domain of CD44 is CD44sR41A.
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. The method according to claim 1, further comprising
administering an additional anti-cancer therapy, wherein the
additional anti-cancer therapy is selected from the group
consisting of surgery, chemotherapy, radiation therapy, targeted
therapy, and immunotherapy.
23-73. (canceled)
74. The pharmaceutical composition of claim 10 further comprising
an additional anti-cancer therapy, wherein the additional
anti-cancer therapy is selected from the group consisting of
surgery, chemotherapy, radiation therapy, targeted therapy, and
immunotherapy.
75. The pharmaceutical composition of claim 74, wherein the
targeted therapy is against the target selected from the group
consisting of EGFR, erbB-2, erbB-3, erbB-4, and c-Met RTK in cancer
cells.
76. The method of claim 22, wherein the targeted therapy is against
the target selected from the group consisting of EGFR, erbB-2,
erbB-3, erbB-4, and c-Met RTK in cancer cells.
77. The method of claim 1, wherein the extracellular domain of CD44
is CD44v3-v10R41A.
78. The method of claim 1, wherein the extracellular domain of CD44
is CD44v8-v10R41A.
79. The method of claim 1, wherein the extracellular domain of CD44
is CD44sR41A.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/274,813, filed Aug. 21, 2009, which is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention is related to pharmaceutical
compositions and methods for the treatment of cancers with CD44
fusion proteins and the derivatives of these fusion proteins. In
certain aspects, these pharmaceutical compositions and methods
include the use of CD44 fusion proteins as single agents and in
combinations with other anti-cancer therapeutics to treat cancers,
including glioma, and to prevent recurrence of cancers, including
that of glioma, after a variety of therapeutic interventions
including surgical removal of cancers. In other aspects, these
pharmaceutical compositions and methods include the use of CD44
fusion proteins along with, prior to, or after other anti-cancer
therapies to treat glioma and other cancer types. CD44 fusion
proteins can be used after other therapeutic interventions as a
maintenance therapy to block expansion of cancer stem cells and to
delay or stop cancer recurrence and metastasis. In another aspect,
the combination of pharmaceutical compositions or methods
administered along with other anti-cancer therapies provides a
synergistic effect on the treatment of glioma and other cancer
types. In other aspects, these pharmaceutical compositions and
methods include the use of CD44 fusion proteins to detect CD44
ligands, including HA, for early cancer diagnosis and prognosis,
and for assessment of patient responses to anti-cancer
treatments.
BACKGROUND OF THE INVENTION
[0004] Conventional anti-cancer therapy is primarily directed at
tumor cell properties that distinguish them from normal cells,
including a generally higher proliferation rate and distinct
metabolic requirements. Although beneficial in a selected group of
malignancies, conventional chemotherapy has a limited effect on the
majority of solid tumors while imposing serious toxicity. More
recent targeted therapeutic strategies are designed to target
specific hyperactivated oncogenes and kinases in cancer cells. They
are generally less toxic than chemotherapy but their efficacies are
limited by tumor cell heterogeneity, ability to switch their
dependence from one aberrant signaling pathway to an alternative
one, and emerging of resistant clones that have acquired new
mutations. It is thus becoming increasingly apparent that merely
targeting cancer cells is unlikely to cure most solid malignancies
(Araujo et al., 2007; Zhang et al., 2009). Accumulating data from
numerous recent observations indicate the host microenvironment
provides an essential contribution to cancer progression, helps
maintain cancer stem cell niches, and modulates the response of
cancer cells to treatment, implying that elements within the tumor
microenvironment may constitute important targets for anti-cancer
therapy (Gilbertson and Rich, 2007; Hideshima et al., 2007;
Hoelzinger et al., 2007; Mantovani et al., 2008; Mishra et al.,
2009; Podar et al., 2009).
[0005] The tumor microenvironment consists of the infiltrating host
cells, including endothelial cells, pericytes, leukocytes, and
fibroblasts, as well as the components of the extracellular matrix
(ECM). Key interactions and cross-talk between tumor cells and
their microenvironment are mediated by their surface receptors
including cell-cell adhesion and ECM receptors that are potentially
attractive therapeutic targets. It has been elegantly shown, for
example, that adhesion of multiple myeloma (MM) cells to the ECM
confers cell adhesion-mediated drug resistance (CAMDR) (Hideshima
et al., 2007). While the molecular basis underlying CAMDR in MM is
still being investigated, interactions between the host
microenvironment and numerous other cancer types along with the
downstream signaling pathways activated by the interactions remain
largely under-explored. Identifying key mediators of tumor cell-ECM
interactions and the corresponding downstream signaling pathway(s)
that promote(s) cancer progression and resistance to therapy are
likely to lead to the development of novel and more efficacious
therapeutic strategies that target cancer cells and their
microenvironment simultaneously.
[0006] Gliomas are the most common type of primary brain cancer and
constitute a spectrum of tumors of variable degrees of
differentiation and malignancy that may arise from the
transformation of neural progenitor cells (Giese et al., 2003;
Maher et al., 2001). The most malignant of these tumors is grade IV
astrocytoma, also known as glioblastoma multiforme (GBM), which
displays highly invasive properties and extremely elevated
chemoresistance. Despite aggressive multimodal therapy, GBM remains
incurable, with an estimated median survival of less than 1 year
and with less than 5% of patients surviving longer than 5 years
(Davis et al., 1998). Identification of novel therapeutic targets,
development of new agents and novel strategies of combinational
treatments to reduce the resistance of GBM to chemo- and
established targeted therapies are therefore urgently needed.
[0007] The central nervous system contains elevated levels of the
broadly distributed glycosaminoglycan hyaluronan (HA); also known
as hyaluronic acid or hyaluronan (Park et al., 2008). Gliomas
express high levels of a major cell surface HA receptor, CD44,
which mediates cell-cell and cell-matrix adhesion and promotes cell
migration and signaling (Stamenkovic and Yu, 2009). CD44 is a
polymorphic cell surface receptor implicated in diverse cellular
functions ((Sherman et al., 1994; Stamenkovic, 2000; Stamenkovic I,
2009; Toole, 2004). It is upregulated in a variety of malignant
tumors and its elevated expression correlates with poor prognosis
of several cancer types (Lim et al., 2008; Matsumura and Tarin,
1992; Pals et al., 1989; Yang et al., 2008). CD44 is believed to
play an important role in the growth and progression of melanoma
(Ahrens et al., 2001; Guo et al., 1994) and breast cancer (Yu and
Stamenkovic, 1999, 2000; Yu et al., 1997) but little is known about
its contribution to the progression of malignant glioma and the
responses of GBM cells and other types of cancer cells to
chemotherapy and targeted therapies.
[0008] CD44 has been shown to be associated with several signaling
components and to serve as a co-receptor with several receptor
tyrosine kinases (RTKs) (Sherman et al., 1994; Stamenkovic, 2000;
Toole, 2004) but no single intact signaling pathway regulated by
CD44 has been defined to date. The cytoplasmic domain of CD44
interacts with members of the Band 4.1 superfamily, including
ezrin-radixin-moesin (ERM) family proteins (Tsukita and Yonemura,
1997) and merlin (Morrison et al., 2001; Sainio et al., 1997),
which serve as linkers between cortical actin filaments and the
plasma membrane and regulate actin cytoskeleton organization and
cell motility (McClatchey and Giovannini, 2005; Okada et al.,
2007). In Drosophila, merlin functions upstream of the Hippo (Hpo)
signaling pathway, which plays an important role in restraining
cell proliferation and promoting apoptosis in differentiating
epithelial cells (Hamaratoglu et al., 2006; Huang et al., 2005;
Pellock et al., 2007). The Drosophila hpo gene encodes a
serine/threonine kinase that phosphorylates and activates the
serine/threonine kinase Warts (Wts). Warts phosphorylates and
inactivates a co-transcription factor Yorkie (Yki), which results
in repression of a common set of downstream target genes, including
dIAP and cyclin E (Hamaratoglu et al., 2006; Huang et al., 2005;
Matallanas et al., 2008; Pellock et al., 2007). Although still
incompletely characterized, the Hippo pathway is believed to be
conserved in mammals where several of its components appear to be
tumor suppressors (Lau et al., 2008; Zeng and Hong, 2008).
Mammalian homologs of Hpo, Wts, Yki, and dIAP are, respectively,
Mammalian Sterile Twenty-like (MST) kinase1 and 2 (MST1/2)
(Lehtinen et al., 2006; Ling et al., 2008; Matallanas et al.,
2008), Large tumor suppressor homolog 1 and 2 (Lats1 and 2) (Hao et
al., 2008; Takahashi et al., 2005), Yes-Associated Protein (YAP)
(Overholtzer et al., 2006), and cellular Inhibitor of Apoptosis
(cIAP1/2) (Srinivasula and Ashwell, 2008). The upstream components
of the mammalian Hippo signaling pathway have not been
identified.
[0009] Efficacies of current available therapies for many malignant
cancers, including glioma, are relative low and render patients
with these diseases poor prognosis with short life expectancy after
the diagnosis. New targets, agents, and combinational therapeutic
approaches for treatment are therefore necessary. In addition, it
would be particularly helpful to be able to that target the bulk of
tumor cells, including glioma, their stem cells, and their
microenvironment simultaneously. The present invention provides
such methods.
SUMMARY OF THE INVENTION
[0010] In certain embodiments of the present invention, a method
for therapeutic intervention or inhibition of cancer recurrence of
a cancer in a mammal is provided, which involves administering to
the mammal in need of such treatment an effective amount of a CD44
fusion protein, which includes the constant region of human IgG1
fused to an extracellular domain of CD44, and wherein the cancer is
glioma, colon cancer, breast cancer, prostate cancer, ovarian
cancer, lung cancer, melanoma, renal cell carcinoma, gastric
cancer, esophageal cancer, pancreatic cancer, liver cancer, or
head-neck cancer.
[0011] In certain aspects of the present invention, a method for
treating a cancer in a mammal is provided, which involves
administering to the mammal in need of such treatment an effective
amount of a CD44 fusion protein, which includes the constant region
of human IgG1 fused to an extracellular domain of CD44, and wherein
the CD44 fusion protein is administered via a virus carrying an
expression vector encoding the CD44 fusion protein and, optionally,
a pharmaceutically acceptable carrier or diluent.
[0012] In certain aspects of the present invention, a method for
treating a cancer in a mammal is provided, which involves
administering to the mammal in need of such treatment an effective
amount of a CD44 fusion protein, which includes the constant region
of human IgG1 fused to an extracellular domain of CD44, and wherein
the CD44 fusion protein is administered as purified protein and b)
a pharmaceutically acceptable carrier or diluent.
[0013] In certain aspects of the present invention, a method for
treating a cancer in a mammal is provided, which involves
administering to the mammal in need of such treatment an effective
amount of a CD44 fusion protein, which includes the constant region
of human IgG1 fused to an extracellular domain of CD44, and wherein
the extracellular domain of CD44 is CD44s, CD44v3-v10, CD44v8-v10,
CD44v4-v10, CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10,
CD44v10, CD44v9, CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3,
CD44sR41A, CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A,
CD44v5-v10R41A, CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A,
CD44v10R41A, CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A,
CD44v5R41A, CD44v4R41A, or CD44v3R41A.
[0014] In other embodiments of the present invention, a
pharmaceutical composition is provided, which includes: a) a CD44
fusion protein comprising the constant region of human IgG1 fused
to an extracellular domain of CD44, wherein the extracellular
domain of CD44 is CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44sR41A,
CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A, CD44v5-v10R41A,
CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A, CD44v10R41A,
CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A, CD44v5R41A,
CD44v4R41A, or CD44v3R41A; and b) a pharmaceutically acceptable
carrier or diluent.
[0015] In other aspects of the present invention, a method for
treating a cancer in a mammal is provided, which involves
administering to the mammal in need of such treatment an effective
amount of a CD44 fusion protein comprising the constant region of
human IgG1 fused to an extracellular domain of CD44 along with one
or more additional anti-cancer therapies.
[0016] In certain aspects of the present invention, the additional
anti-cancer therapies are surgery, chemotherapy, radiation therapy,
targeted therapy, and immunotherapy. In certain aspects of the
present invention, the additional anti-cancer therapy is radiation
therapy. In other aspects of the present invention, the additional
anti-cancer therapy is surgery.
[0017] In certain aspects of the present invention, the additional
anti-cancer therapy is chemotherapy. In certain aspects of the
present invention, the chemotherapy is temozolomide, carmustine,
docetaxel, carboplatin, cisplatin, epirubicin, oxaliplatin,
cyclophosphamide, methotrexate, fluorouracil, vinblastine,
vincristine, mitoxantrone, satraplatin, ixabepilone, pacitaxel,
gemcitabine, capecitabine, doxorubicin, etoposide, melphalan,
hexamethylamine, irinotecan, or topotecan. In another aspect of the
present invention, the chemotherapy is temozolomide or
carmustine.
[0018] In certain aspects of the present invention, the additional
anti-cancer therapy is targeted therapy. In certain embodiment of
the present invention, the targeted therapy is a receptor tyrosine
inhibitor. In certain embodiment of the present invention, the
targeted therapy is an inhibitor of erbB receptor. In certain
embodiment of the present invention, the targeted therapy is an
inhibitor of c-Met. In certain embodiment of the present invention,
the targeted therapy is an inhibitor of VEGFR. In certain
embodiment of the present invention, the targeted therapy is an
agent that promotes apoptosis and stress responses. In certain
embodiment of the present invention, the targeted therapy is an
inhibitor of the Wnt signaling pathway. In certain embodiments of
the present invention, the targeted therapy is Trastuzumab,
cetuximab, panitumumab, gefitinib, erlotinib, lapatinib, BIBW2992,
CI-1033, PF-2341066, PF-04217903, AMG 208, JNJ-38877605, MGCD-265,
SGX-523, GSK1363089, sunitinib, sorafenib, vandetanib, BIBF1120,
pazopanib, bevacizumab, vatalanib, axitinib, E7080, perifosine,
MK-2206, temsirolimus, rapamycin, BEZ235, GDC-0941, PLX-4032,
imatinib, AZD0530, bortezomib, XAV-939, advexin (Ad5CMV-p53),
Genentech--Compound 8/cIAP-XIAP inhibitor, or Abbott
Laboratories--Compound 11.
[0019] In other embodiments of the present invention, a
pharmaceutical composition is provided, which includes: a) a CD44
fusion protein comprising the constant region of human IgG1 fused
to an extracellular domain of CD44, wherein the extracellular
domain of CD44 is a CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44sR41A,
CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A, CD44v5-v10R41A,
CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A, CD44v10R41A,
CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A, CD44v5R41A,
CD44v4R41A, or CD44v3R41A; b) at least one therapeutic agent that
causes cytotoxic or cytostatic stress in cancer cells; and c) a
pharmaceutically acceptable carrier or diluent.
[0020] In another embodiments of the present invention, a
pharmaceutical composition is provided, which includes: a) a CD44
fusion protein comprising the constant region of human IgG1 fused
to an extracellular domain of CD44, wherein the extracellular
domain of CD44 is a CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44sR41A,
CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A, CD44v5-v10R41A,
CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A, CD44v10R41A,
CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A, CD44v5R41A,
CD44v4R41A, or CD44v3R41A; b) at least one therapeutic agent that
inhibits EGFR/erbB-2/erbB-3/erbB-4/c-Met/VEGFR RTK in cancer cells;
and c) a pharmaceutically acceptable carrier or diluent. In certain
aspects of the present invention, the inhibitor of
EGFR/erbB-2/erbB-4/c-Met/VEGFR RTK includes Trastuzumab, cetuximab,
panitumumab, gefitinib, erlotinib, lapatinib, BIBW2992, CI-1033,
PF-2341066, PF-04217903, AMG 208, JNJ-38877605, MGCD-265, SGX-523,
GSK1363089, sunitinib, sorafenib, vandetanib, BIBF1120, pazopanib,
bevacizumab, vatalanib, axitinib, and E7080.
[0021] In another embodiments of the present invention, a
pharmaceutical composition is provided, which includes: a) a CD44
fusion protein comprising the constant region of human IgG1 fused
to an extracellular domain of CD44, wherein the extracellular
domain of CD44 is a CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44sR41A,
CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A, CD44v5-v10R41A,
CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A, CD44v10R41A,
CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A, CD44v5R41A,
CD44v4R41A, or CD44v3R41A; b) at least one therapeutic agent that
inhibits IAPs or promotes stresses in cancer cells; and c) a
pharmaceutically acceptable carrier or diluent. In certain aspects
of the present invention, the inhibitor of IAPB or promotes
stresses includes advexin (Ad5CMV-p53), Genentech--Compound
8/cIAP-XIAP inhibitor, Abbott Laboratories--Compound 11,
perifosine, MK-2206, temsirolimus, rapamycin, BEZ235, GDC-0941,
PLX-4032, imatinib, AZD0530, bortezomib, or XAV-939.
[0022] In other embodiments of the present invention, a
pharmaceutical composition is provided, which includes: a) a virus
carrying a expression vector encoding a CD44 fusion protein
comprising the constant region of human IgG1 fused to an
extracellular domain of CD44, wherein the extracellular domain of
CD44 is CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10, CD44v5-v10,
CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9, CD44v8,
CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44sR41A, CD44v3-v10R41A,
CD44v8-v10R41A, CD44v4-v10R41A, CD44v5-v10R41A, CD44v6-v10R41A,
CD44v7-v10R41A, CD44v9-v10R41A, CD44v10R41A, CD44v9R41A,
CD44v8R41A, CD44v7R41A, CD44v6R41A, CD44v5R41A, CD44v4R41A, or
CD44v3R41A; and b) a pharmaceutically acceptable carrier or
diluent.
[0023] In certain aspects of the above embodiments, the cancer is
glioma, colon cancer, breast cancer, prostate cancer, ovarian
cancer, lung cancer, melanoma, renal cell carcinoma, gastric
cancer, esophageal cancer, pancreatic cancer, liver cancer or
head-neck cancer. In certain embodiments of the present invention
the glioma is an astrocytoma. In other embodiments of the present
invention the glioma is a glioblastoma multiforme. In certain
embodiments of the present invention the mammal is a human.
[0024] In certain aspects of the above embodiments, the
extracellular domain of the CD44 is CD44v3-v10. In other aspects of
the above embodiments, the extracellular domain of the CD44 is
CD44v8-v10. In another aspect of the above embodiments, the
extracellular domain of the CD44 is CD44s. In another aspect of the
above embodiments, the extracellular domain of the CD44 is
CD44v6-v10.
[0025] In other embodiments of the present invention, methods of
detecting hyaluronan in a sample are provided, comprising
contacting the sample with a labeled CD44 fusion protein comprising
the constant region of human IgG1 fused to an extracellular domain
of CD44. In certain embodiments, the sample is a cancer biopsy or
cancer section. In other embodiments, the sample is a patient fluid
sample is blood, serum, plasma, or urine. In other embodiments, the
label is biotin, fluorescent labels, alkaline phosphatase,
horseradish peroxidase, magnetic beads, or radioactive labels. In
yet another embodiment, the methods further comprise incubating the
labeled CD44 fusion protein in the sample and quantifying the label
bound to hyaluronan. In yet another embodiment, the extracellular
domain of CD44 is CD44v3-v10, CD44v8-v10, CD44s, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, or CD44v3.
[0026] In other embodiments of the present invention, methods of
diagnosing a cancer in a mammal are provided, comprising detecting
hyaluronan in a sample from the mammal, wherein the detecting is
done according to any of the described methods of detecting
hyaluronan, and wherein an increase in the amount of hyaluronan in
the sample compared to a normal control sample indicates the
presence of cancer. In certain embodiments, the cancer is a glioma,
colon cancer, breast cancer, prostate cancer, ovarian cancer, lung
cancer, renal cell carcinoma, gastric cancer, esophageal cancer,
head-neck cancer, pancreatic cancer, or melanoma. In certain other
embodiments, the methods further comprise detecting CD44 in the
sample, and wherein an increase in the amount of hyaluronan and
CD44 in the sample compared to a normal control sample indicates
the presence of cancer.
[0027] In other embodiments of the present invention, methods of
determining a change in the cancerous state of a mammal are
provided, comprising collecting a first sample from the mammal,
detecting hyaluronan in the first sample from the mammal, wherein
the detecting is done according to any of the described methods of
detecting hyaluronan, collecting a second sample from the mammal,
detecting hyaluronan in a second sample from the mammal, wherein
the detecting is done according to any of the described methods of
detecting hyaluronan, wherein a difference in the amount of
hyaluronan in the second sample compared to the amount in the first
sample indicates a change in the cancerous state of the mammal.
[0028] These and others aspects of the present invention will be
apparent to those of ordinary skill in the art in light of the
present specification, claims, and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A: Bar graph showing the up-regulation of expression
of CD44 transcripts in microarray data sets (derived from
http://www.oncomine.org/) of multiple glioma tissues compared to
normal human brain tissues (study 1, 2, and 4) or to normal white
matter (study 3). CD44 expression in brains or white matter from
epilepsy patients (bars on the left of each study) or in GBMs (bars
on the right of each study) are shown.
[0030] FIG. 1B: Representative pictures of CD44
immunohistochemistry performed on 14 GBM tissues (B-a) and 8 normal
human brain samples (B-b) using anti-CD44 antibody (Santa Cruz).
Bar, 50 .mu.m.
[0031] FIG. 1C: Western Blot of endogenous CD44 expression in a
panel of human glioma cells using anti-CD44 antibody (Santa Cruz).
Proteins from normal human astrocytes (NHAs, ALLCELLS, Inc.) were
loaded in lanes 1 and 20. Actin was included as an internal control
for loading (lower panels). The molecular weight bars correspond to
197 kDa, 110 kDa, and 72 kDa.
[0032] FIG. 2A: Western blot of CD44 expression knockdown by
lentiviral based shRNAs in U251 (A-a) or U87MG (A-b) cells using
anti-CD44 mAb (Santa Cruz).
[0033] FIG. 2B: Immunocytochemistry shows reduced endogenous CD44
levels in U87MG cells infected with different CD44 knockdown shRNA
lentiviral vectors (b-c) comparing to U87MG cells infected with
non-targeting (TRC-NT) control shRNA (a) using anti-CD44 mAb (Santa
Cruz).
[0034] FIG. 2C: Fluorescent-HA (FL-HA) binding assay with wild type
U87MG cells (C-a), U87MG cells infected with TRC-NT control shRNA
(C-c), and U87MG cells infected with different CD44 knockdown
shRNAs lentiviral vectors (C-b, -d) as indicated in the panels.
[0035] FIG. 2D: Immunocytochemistry of endogenous CD44 levels in
U251 cells infected with different CD44 knockdown shRNA
lentiviruses (a-d) or TRC-NT control shRNA lentiviruses (e) using
anti-CD44 mAb (Santa Cruz).
[0036] FIG. 3A: Bar graph showing that knockdown of CD44 expression
inhibits subcutaneous growth of U87MG glioma cells in vivo.
[0037] FIG. 3B: Bar graph showing that knockdown of CD44 expression
inhibits subcutaneous growth of U251 glioma cells in vivo.
[0038] FIG. 3C: Line graph showing the in vivo growth rates of the
subcutaneous tumors derived from U87MG cells infected with
different shRNA constructs.
[0039] FIG. 3D: Line graph showing the in vivo growth rates of the
subcutaneous tumors derived from U251 cells infected with different
shRNA constructs.
[0040] FIG. 3E: Morphology (H&E), proliferation (Brdu and Ki67)
and apoptosis (Apoptag) status of subcutaneously explanted glioma
tumors.
[0041] FIG. 4A: Bioluminescence imaging analysis of mice 3, 6, 9,
and 13 days following the intracranial injection of U87MG-TRC-NT,
U87MGshRNAmir-NT (non-targeting shRNA controls, upper panels), and
U87MG-TRC-CD44#3 and U87MGshRNAmir-CD44#1 (shRNAs against human
CD44, bottom panels).
[0042] FIG. 4B: Line graph showing the survival rates of mice
following intracranial injections of the transduced U87MG (B-a) and
U251 (B-b) cells as detailed in the panels.
[0043] FIG. 4C: Bioluminescence imaging analysis of mice 6, 9, 13,
and 17 days following intracranial injection of U87MG-NT (U87MG
cells infected with a mixture of lentiviruses carrying
non-targeting TRC-NT and shRNAmir-NT constructs), U87MGshRNA-CD44
(U87MG cells infected with a mixture of lentiviruses carrying
TRC-CD44#3 and shRNAmir-CD44#1 constructs, which effectively knock
down CD44 expression). These mice were treated with or without
chemotherapeutic agents (BCNU or TMZ) as detailed in the
panels.
[0044] FIG. 4D: Line graph showing the survival rates of mice
following intracranial injections of the transduced U87MG (D-a) and
U251 (D-b) cells with or without CD44 knockdown. These mice were
treated with or without chemotherapeutic agents (BCNU or TMZ) as
detailed in the panels.
[0045] FIG. 5A: Western blot analysis of the levels of
phosphorylated and/or total merlin, MST1/2, Lats1/2, YAP, cIAP1/2,
and cleaved caspase 3 induced by oxidative stresses
(H.sub.2O.sub.2) in U87MG cells infected with a mixture of
lentiviruses carrying non-targeting TRC-NT and shRNAmir-NT
constructs.
[0046] FIG. 5B: Western blot analysis of the levels of
phosphorylated and/or total merlin, MST1/2, Lats1/2, YAP, cIAP1/2,
and cleaved caspase 3 induced by oxidative stresses in U87MG cells
infected with a mixture of lentiviruses carrying shRNAs against
human CD44, TRC-CD44#3 and shRNAmir-CD44#1, which resulted in
effective knockdown of CD44 in these cells.
[0047] FIG. 5C: Western blot analysis of the levels of
phosphorylated and/or total JNK and p38 stress kinases, p53, p21,
and puma induced by oxidative stresses in U87MG cells infected with
a mixture of lentiviruses carrying non-targeting TRC-NT and
shRNAmir-NT constructs.
[0048] FIG. 5D: Western blot analysis of the levels of
phosphorylated and/or total JNK and p38 stress kinases, p53, p21,
and puma in U87MG cells infected with a mixture of lentiviruses
carrying shRNAs against human CD44, TRC-CD44#3 and
shRNAmir-CD44#1.
[0049] FIG. 6A: Western blot analysis of the levels of
phosphorylated and/or total MST1/2, YAP, cIAP1/2, JNK and p38
stress kinases, p53, and p21 induced by a chemotherapeutic agent,
TMZ, in U87MG cells infected with a mixture of lentiviruses
carrying non-targeting TRC-NT and shRNAmir-NT constructs.
[0050] FIG. 6B: Western blot analysis of the levels of
phosphorylated and/or total MST1/2, YAP, cIAP1/2, JNK and p38
stress kinases, p53, and p21 induced by a chemotherapeutic agent,
TMZ, in U87MG cells infected with a mixture of lentiviruses
carrying shRNAs against human CD44, TRC-CD44#3 and
shRNAmir-CD44#1.
[0051] FIG. 7A: Western blot of the activation of Erk1/2 kinases
induced by the ligands of erbB and c-Met receptor tyrosine kinase
(RTK) in U87MG cells infected with a mixture of lentiviruses
carrying non-targeting TRC-NT and shRNAmir-NT constructs.
[0052] FIG. 7B: Western blot of the activation of Erk1/2 kinases
induced by the ligands of erbB and c-Met receptor tyrosine kinase
(RTK) in U87MG cells infected with a mixture lentiviruses carrying
shRNAs against human CD44, TRC-CD44#3 and shRNAmir-CD44#1.
[0053] FIG. 8. The signaling pathways that are significantly
affected by increased expression of merlin, the downstream CD44
effector that is negatively regulated by CD44, in U87MG human
glioma and WM793 human melanoma cells. Functional analysis of the
data sets from microarray experiments is shown. The data indicates
that increased expression of merlin activates Hippo and inhibits
Wnt and c-Met signaling pathways.
[0054] FIG. 9. Merlin inhibits canonical Wnt signaling in human
glioma cells. A-B, Luciferase activity was measured in U87MGwt,
U87MGmerlin, U87MGmerlinS518D, and U87MGmerlinS518A cells 24 hours
after transfection of cells with TopFlash (A) or FopFlash (B) in
triplicates.
[0055] FIG. 10. A model of merlin-mediated signaling events and
their potential cross-talk. The components of Drosophila Hippo
signaling pathway are underlined. Merlin functions upstream of the
mammalian Hippo (merlin-MST1/2-LATS1/2-YAP) and JNK/p38 signaling
pathways and plays an essential role in regulating the cell
response to the stresses and stress-induced apoptosis as well as to
proliferation/survival signals. CD44 functions upstream of merlin
and Hippo signaling pathway. Merlin and CD44 antagonize each
other's function. Merlin inhibits activities of RTKs and the
RTK-derived growth and survival signals. CD44 function upstream of
mammalian Hippo signaling pathway and enhances activities of RTKs
and Wnt signaling.
[0056] FIG. 11. Biochemical and functional properties of hsCD44-Fc
and hsCD44R41A-Fc fusion proteins. A, Western blot analysis of
serum-free cell culture supernatants derived from U251 cells
transduced with retroviruses carrying the expression constructs of
hsCD44s-Fc, hsCD44v8-v10-Fc, hsCD44v3-v10-Fc, hsCD44sR41A-Fc,
hsCD44v8-v10R41A-Fc, hsCD44v3-v10R41A-Fc, or the empty expression
vector. Anti-CD44 antibody (Santa Cruz) was used to detect the
fusion proteins. The molecular weight bars correspond to 199 kDa
and 116 kDa. B, FL-HA binding assays were performed. U251 cells
expressing hsCD44s-Fc, hsCD44sR41A-Fc, or infected with
retroviruses carrying the empty expression vectors were cultured
for two days. FL-HA (20 .mu.g/ml) was added into culture media and
the cells were cultured additional 12 h before fixing the cells.
Bar, 40 .mu.m. C, hsCD44v3-v10-Fc proteins are modified by heparan
sulfate (HS). Purified hsCD44s-Fc, hsCD44v8-v10-Fc, and
hsCD44v3-v10-Fc fusion proteins were treated with or without
heparinase VIII before eluting from protein A columns. These
proteins were bound onto Elias plates in triplicate. After blocking
with BSA, the coated proteins reacted with anti-HS antibody
(Calbiochem). The intensity of the reaction color was measure by an
Elisa reader and normalized by the reactivity to anti-CD44
antibody, which provides relative quantity of the coated fusion
proteins on the plates.
[0057] FIG. 12. Antagonists of CD44 are effective therapeutic
agents against human GBM in mouse models. A, U87MG and U251 cells
were transduced with the retroviruses carrying empty vector and the
expression constructs of hsCD44s-Fc, hsCD44v8-v10-Fc, and
hsCD44v3-v10 and their serum free cell culture supernatants were
collected and analyzed on Western blots using anti-CD44 antibody
(Santa Cruz) or anti-human IgG antibody. The molecular weight bars
correspond to 199 kDa and 116 kDa. B, 2.times.10.sup.6 of the
transduced U87MG and U251 cells were injected subcutaneously per
mouse. Growth rates of the subcutaneous tumors were determined and
expressed as the mean of tumor volume (mm.sup.3)+/-SD. Six mice
were used for each construct. C, Survival rates of mice following
the intracranial injections of transduced U87MG (C-a) and U251
(C-b-c) cells infected with the retroviruses carrying the empty
expression vectors, hsCD44s-Fc, hsCD44v8-v10-Fc, hsCD44v3-v10,
hsCD44sR41A-Fc, or hsCD44v3-v10R41A-Fc constructs as indicated in
the panels. 15 mice were used for each type of transduced glioma
cells in a-b and ten mice were used in panel c.
[0058] FIG. 13. Purified hsCD44s-Fc fusion proteins inhibit
intracranial glioma growth in Rag-1 mice and display an intra-tumor
distribution pattern without apparent toxicity. A-B, Treatment of
pre-established intracranial U87MG (A) and U251 (B) gliomas with
intravenous delivery of 5 mg/kg purified hsCD44s-Fc fusion proteins
or human IgG every third day. The results show that hsCD44s-Fc but
not human IgG significantly extended the survival of the
experimental mice (p<0.001). Six Rag-1 mice were used for each
treatment. C, Distribution of hsCD44s-Fc fusion proteins in
intracranial gliomas (C-b and C-d) and normal adjacent brain
tissues (C-a, and C-c). The fusion proteins were detected by
anti-human IgG antibody. Bar in a and b, 100 .mu.m and in c and d,
50 .mu.m. D, Purified CD44s-Fc fusion protein displayed no apparent
toxicity towards normal host tissues. H&E staining of normal
tissues derived from the Rag-1 mice received iv injection of 5
mg/kg of hsCD44s-Fc fusion proteins (right side panels) or human
IgG (left side panels) every third day. Bar, 50 .mu.m.
[0059] FIG. 14. A. Glioma cell viability assays: knockdown of human
CD44 sensitizes the response of GBM cells to a dual EGFR/erbB-2
inhibitor, BIBW2992. B. Glioma cell viability assays: knockdown of
human CD44 sensitizes the response of GBM cells to a pan
EGFR/erbB-2/erbB-4 inhibitor, CI-1033. C. Glioma cell viability
assays: knockdown of human CD44 sensitizes the response of GBM
cells to a c-Met inhibitor, SU11274.
[0060] FIG. 15. hsCD44-Fc fusion proteins sensitize the responses
of GBM cells to chemotherapeutic and targeted agents. Glioma cell
viability assays were performed using the Cell Titer-Glo
Luminescent Cell Viability Assay kit (Promega) following
manufacturer's instruction. U87MG cells were plated in triplicate
at 1.times.10.sup.5 cells per well treated different concentrations
of TMZ (A), gefitinib (B), BIBW2992 (C), CI-1033 (D), or PF-2341066
(E) as detailed in the panels in the presence or absence of
hsCD44s-Fc fusion proteins or human IgG (10 .mu.g/ml) for 48 hours
before the cell viability was measured.
[0061] FIG. 16. hsCD44s-Fc fusion protein displayed low
cytotoxicity toward a panel of normal cells. Cell viability assays
were performed as described in FIG. 15. NHAs, normal human Schwann
cells, HUVECs, normal fibroblasts, and U251 GBM cells were plated
in triplicate at 1.times.10.sup.5 cells/well in 96 well plates and
treated different concentrations of purified hsCD44s-Fc fusion
proteins for 48 hours before the cell viability was measured.
[0062] FIG. 17. Establishment and characterization of primary human
glioma spheres (GBM stem cells, GBMCSCs) from fresh GBM tissues. A,
Self-renewal capacity of glioma spheres. A-a, single primary
GBMCSCs were maintained in serum free stem cell medium (SCCM).
Small (b), intermediate (c), and large (d) GBM spheres were formed
after culturing for 2-3, 4-5, and 6-7 days in SCCM. B, The glioma
spheres were disaggregated and cultured in SCCM for 12 hours and
stained positive for the glioma stem cell markers, nestin (B-a) or
Sox2 (B-b). Another set of the cells were cultured in astrocyte
medium (ScienCell) for 6 day before stained positive for a
differentiated astrocyte specific marker, glial fibrillary acidic
protein (GFAP, B-c). In panel B-d, only second antibody was used as
a control showing the absence of non-specific staining. Bar, 150
.mu.m. C, human glioma spheres (HGSs), MSSM-GBMCSC-1, were
disaggregated and seeded on the BD BioCoat.TM. Matrigel.TM. Matrix
6-well plates, which were designed to maintain and propagate
embryonic stem cells in the absence of feeder layers. These cells
were transduced with retroviruses carrying GFP. After selection
with puromycin, the pooled populations of drug-resistant cells were
suspended into single cells and culture in SCCM in ultra-low
attachment plates to re-form spheres. GFP expression by the
re-formed spheres (C-a), morphology (C-b) and merged pictures (C-c)
of these spheres are shown. Bar: 300 .mu.m. D, MSSM-GBMCSC-1 cells
form invasive intracranial tumors .about.25 days after injection of
5.times.10.sup.4 of the cells (D-a) and overexpression of
hsCD44s-Fc fusion proteins inhibits intracranial growth of
MSSM-GBMCSC-1 cells (D-b).
[0063] FIG. 18. Morphology of glioma sphere cells, MSSM-GBMCSC-1,
derived from a GBM patient showing that knockdown of CD44
expression by a mixture of lentiviruses carrying shRNAs against
human CD44, TRC-CD44#3 and shRNAmir-CD44#1, inhibits the formation
of glioma spheres.
[0064] FIG. 19. CD44 expression. A: Bar graph showing that CD44
mRNA expression level is up-regulated in human colon cancer (right
side of each study) comparing to normal colon (left side of each
study) (data derived from http://www.oncomine.org/). B:
Representative pictures of CD44 immunohistochemistry performed on 6
normal colon tissue samples (B-a), 6 malignant colon cancer samples
(B-b), and 6 liver metastasis of colon cancers (B-c) using
anti-CD44 antibody (Santa Cruz). C: Additional bar graphs showing
that CD44 mRNA expression level is up-regulated in human colon
cancer (right side of each study) comparing to normal colon (left
side of each study) (data derived from
http://www.oncomine.org/).
[0065] FIG. 20. A: Western blot of CD44 expression knockdown in
HCT116 human colon cancer cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of HCT116 human colon
cancer cells in vivo. HCT116 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs. (n=6)
[0066] FIG. 21A. Western blot of CD44 expression knockdown in
KM20L2 human colon cancer cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). 21B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of KM20L2 human colon
cancer cells in vivo. KM20L2 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs. (n=6)
[0067] FIG. 22. Representative pictures of CD44
immunohistochemistry performed on 6 normal prostate tissue samples
(A) and 6 malignant prostate cancer samples (B) using anti-CD44
antibody (Santa Cruz), showing that CD44 is up-regulated in
malignant prostate cancer.
[0068] FIG. 23. A: Western blot of CD44 expression knockdown in
PC3/M human prostate carcinoma cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). 23B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of PC3/M human prostate
carcinoma cells in vivo. PC3/M cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs (n=6).
[0069] FIG. 24. CD44-Fc fusion proteins are effective therapeutic
agents against human prostate cancer cells in vivo. A, expression
of CD44 by human prostate cancer cells were assessed by Western
blotting using anti-CD44 antibody (Santa Cruz). B, 5.times.10.sup.6
PC3/M cells were injected subcutaneously into each Rag-1 mice. The
tumors were allowed to grow for .about.two weeks when tumor volumes
reach .about.150 mm.sup.3. The mice bearing similar size tumors
were separated into 6 groups (6mice/group) and treated every other
days with 4 intratumoral injections of 5 .mu.l/injection of 10
mg/ml of hsCD44s-Fc, hsCD44v8-v10-Fc, hsCD44v6-v10-Fc,
hsCD44v3-v10-Fc, or human IgG, or 0.9% NaCl. The experiments were
stopped when tumors of control groups (treatment of human IgG or
0.9% NaCl) reached .about.1 cm in their longest diameters. All the
tumors were dissected out and weighted. Data is presented as the
mean of tumor weight+/-SD.
[0070] FIG. 25. Human malignant breast cancer cells that
infiltrated host stroma expresses a high level of CD44 and breast
cancer stroma accumulates a high level of hyaluronan (HA).
Expression of CD44 protein (A-C) is up-regulated in malignant
breast cancer cells that infiltrated stroma (B-C) compared to
normal human breast epithelia (A) as assessed by
immunohistochemistry using anti-CD44 antibody (Santa Cruz). In
addition, a much higher level of HA is accumulated in breast cancer
stroma (E) compared to normal breast stroma (D). HA was detected by
biotinylated hsCD44-Fc fusion proteins. Representative images from
6 normal and 6 malignant breast cancer tissues are shown. Bar in A,
C-E, 50 .mu.m and in B, 200 .mu.m.
[0071] FIG. 26A: Western blot of CD44 expression knockdown in MX-2
human breast carcinoma cells transduced with shRNAs against human
CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody (Santa
Cruz). 26B: Bar graph showing that knockdown of CD44 expression
inhibits subcutaneous growth of MX-2 human breast carcinoma cells
in vivo. MX-2 cells were transduced with shRNAs against human CD44
or non-targeting (NT) shRNAs (n=6).
[0072] FIG. 27. A: Western blot of CD44 expression knockdown in
SW613 human breast carcinoma cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of SW613 human breast
carcinoma cells in vivo. SW613 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs (n=6).
[0073] FIG. 28. Establishment and characterization of human breast
cancer stem cells (BCSCs) and in vivo xenograft breast cancer
models. A, CD44 expression by normal human mammary epithelial cells
(line 1), MSSM-BCSC-1, -2, and -3 (lane 2-4) was determined by
western blots using anti-CD44 mAb. Actin levels were used as
loading controls (the bottom panel). B, The BCSCs express high
levels of the stem cells markers as assessed by immunocytochemistry
using antibodies against CD44, Sox-2, Oct3/4, and SSEA1 and they
express a low level of CD24. Bar, 100 .mu.m. C-a-c, Self-renewal
capacity of the mammospheres. C-a, single primary BCSCs were
maintained in serum free stem cell medium (SCCM). Intermediate
(C-b) and large (C-c) mammospheres were formed after culturing for
4-5, and 6-7 days in SCCM. C-d-f, Morphology of mammospheres
showing that knockdown of CD44 expression in BCSCs inhibits the
sphere formation (f) whereas non-targeting shRNAs have no effect
(e) when compared to the parental BCSCs (d). Bar, 200 .mu.m. D,
Quantitative analysis revealed the inhibitory effect of CD44
knockdown on the sphere formation. The numbers of spheres were
counted in ten randomly selected 100.times. microscopic field,
averaged, and presented as the means+/-SD. E, Bioluminescence
images of subcutaneous tumors derived from MSSM-BCSCs.
[0074] FIG. 29. Antagonists of CD44, hsCD44v3-v10-Fc,
hsCD44v6-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc fusion proteins,
are effective therapeutic agents against human breast cancer stem
cells in vivo. A, analysis of purified hsCD44s-Fc, hsCD44v8-v10-Fc,
hsCD44v6-v10, and hsCD44v3-v10 by Western blotting using anti-CD44
antibody (Santa Cruz). B, 1.times.10.sup.6 MSSM-BCSC-1 cells were
injected subcutaneously into each Rag-1 mice. The tumors were
allowed to growth for .about.three weeks when the tumor volumes
reach .about.200 mm.sup.3. The mice bearing similar size tumors
were separated into 6 groups (6mice/group) and were treated every
other days with 4 intratumoral injections of 5 .mu.l/injection of
10 mg/ml of hsCD44s-Fc, hsCD44v8-v10-Fc, hsCD44v6-v10-Fc,
hsCD44v3-v10-Fc, or human IgG, or 0.9% NaCl. The experiments were
stopped when tumors of the control groups (treatment of human IgG
or 0.9% NaCl) reached .about.1 cm in their longest diameters. All
the tumors were dissected out and weighted. Data is presented as
the mean of tumor weight+/-SD.
[0075] FIG. 30. A: Western blot of CD44 expression knockdown in
NCI-H125 human lung cancer cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of NCI-H125 human lung
cancer cells in vivo. NCI-H125 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs (n=6).
[0076] FIG. 31. A: Western blot of CD44 expression knockdown in
NCI-H460 human lung cancer cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of NCI-H460 human lung
cancer cells in vivo. NCI-H460 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs. (n=6)
[0077] FIG. 32. A: Bar graph showing that CD44 mRNA expression
level is up-regulated in human ovarian cancer (right side of each
study) comparing to normal ovary (left side of each study) (data
derived from http://www.oncomine.org/). B-D. Stroma of stage III/IV
ovarian cancer tissues express high levels of CD44 (C) and/or
hyaluronan (HA, D) compared to normal ovary (B). Levels of CD44
protein and HA were assessed by immunohistochemistry using
anti-CD44 antibody (Santa Cruz, B and C) and biotinylated hsCD44-Fc
(D), respectively. OSE: ovary surface epithelial cells. Bar, 50
.mu.m in C and 100 .mu.m in A-B.
[0078] FIG. 33. A: Western blot of CD44 expression knockdown in
OVCAR-3 human ovarian cancer cells transduced with shRNAs against
human CD44 or non-targeting (NT) shRNAs using anti-CD44 antibody
(Santa Cruz). B: Bar graph showing that knockdown of CD44
expression inhibits subcutaneous growth of OVCAR-3 human ovarian
cancer cells in vivo. OVCAR-3 cells were transduced with shRNAs
against human CD44 or non-targeting (NT) shRNAs (n=6).
[0079] FIG. 34. Establishment of ovarian cancer stem cells (OCSCs,
B-C, E) and in vivo ascites tumor models (A). B, Positive
expression of stem cell markers are shown as assessed by
immunocytochemistry using antibodies against CD44 (B-a), Sox-2
(B-b), Oct3/4 (B-c), and Nanog (B-d). Bar, 50 .mu.m. C, Formation
of ascites tumors in Rag-1 mice by MSSM-OCSC1 cells: MSSM-OCSC1
cells formed tumors that attached to peritoneal wall (C-a), liver
(C-b), and mesentery (C-c). Bar, 150 .mu.m. D, Western blot
analysis of CD44 expression in MSSM-OCSC1 cells transduced with
shRNAs against CD44 (lane 3) or non-targeting shRNAs (lane 2). CD44
level in parental cells is shown in lane 1. MSSM-OCSCs express
several CD44 variants and the standard form of CD44, CD44s (the
lower band). E-a-c, Self-renewal capacity of MSSM-OCSC-1 spheres.
The suspended OCSCs were maintained in serum free stem cell medium
for 2-3 (a), 4-5 (b), and 6-7 (c) days. E-d-f, CD44 is required for
self-renewal of OCSCs: Knockdown of CD44 (D-f) but not parental
(D-d) or non-targeting shRNA (D-e) inhibits the formation of OCSC
spheres. F, Quantitative analyses of D-d-f are shown. The numbers
of spheres in twenty randomly selected 100.times. microscopic
fields were counted, averaged, and presented as the means+/-SD.
[0080] FIG. 35. CD44 expression is up-regulated in human melanomas
(A-B) and melanoma cells (C). A-B, CD44 mRNA levels in Talantov
Melanoma data set (www.oncomine.org). C, CD44 expression in human
melanocytes and melanoma cells was assessed by Western blotting
using anti-CD44 antibody.
[0081] FIG. 36. hsCD44-Fc fusion proteins inhibit human melanoma
growth in vivo. A, Overexpression of hsCD44s-Fc, hsCD44v8-v10-Fc,
and hsCD44v3-v10-Fc fusion proteins by human M14 melanoma cells
(lane 1-3). B, 5.times.10.sup.6 of M14 cells expressing different
CD44-Fc fusion proteins or transduced with empty expression vectors
were injected subcutaneously into each Rag-1 mice. Tumors were
allowed to grow for .about.four weeks. At the end of experiments,
all the tumors were dissected out and weighted. Data is presented
as the mean of tumor weight (gram)+/-SD.
[0082] FIG. 37. CD44 mRNA level is up-regulated in human clear cell
sarcoma and renal cell carcinoma (right side of the studies)
comparing to normal fetal kidney (left side of the studies). Data
derived from oncomine (www.oncomine.org).
[0083] FIG. 38. CD44 mRNA level is up-regulated in human Head and
Neck Squamous carcinoma (A, right side of the study) and renal cell
carcinoma (B, right side of the study comparing to normal oral
mucosa (left side of A) or normal kidney tissues (left side of B).
Data derived from oncomine (www.oncomine.org).
[0084] FIG. 39. CD44 mRNA level is up-regulated in human oral
cavity carcinoma (left panel), head and neck squamous cell
carcinoma (the middle panel), and tongue squamous cell carcinoma
when compared to their normal counterparts. Data derived from
oncomine (www.oncomine.org).
[0085] FIG. 40. hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc
inhibits subcutaneous growth of human head and neck cancer cells in
vivo. A, expression of CD44 by human head and neck carcinoma cells
were assessed by Western blotting using anti-CD44 antibody (Santa
Cruz). Expression level of CD44 by these carcinoma cells correlates
with their tumorigenicity in vivo. B, overexpression of hsCD44s-Fc,
hsCD44v8-v10-Fc, and hsCD44v3-v10-Fc fusion proteins by SCC-4
head-neck carcinoma cells. C, 5.times.10.sup.6 SCC-4 cells
expressing different CD44-Fc fusion proteins or transduced with
empty expression vectors were injected subcutaneously into each
Rag-1 mice. Tumors were allowed to grow for .about.two months. At
the end of experiments, all the tumors were dissected out and
weighted. Data is presented as the mean of tumor weight+/-SD.
[0086] FIG. 41. A, Expression of CD44 by human pancreatic cancer
cells (BXPC-3, PAN-08-13, PAN-08-27, and PAN-10-05) and a human
hepatocellular carcinoma cell line (SK-Hep-1). B, overexpression of
hsCD44s-Fc, hsCD44v8-v10-Fc, and hsCD44v3-v10-Fc fusion proteins by
human BXPC-3 pancreatic cells (lane 1-3) or human SK-Hep-1
hepatocellular carcinoma cells (lane 4-6). C, 5.times.10.sup.6
BXPC-3 (C-a) or SH-Hep-1 (C-b) cells expressing different CD44-Fc
fusion proteins or transduced with empty expression vectors were
injected subcutaneously into each Rag-1 mice. Tumors were allowed
to grow for .about.five-six weeks. At the end of experiments, all
the tumors were dissected out and weighted. Data is presented as
the mean of tumor weight (gram)+/-SD.
[0087] FIG. 42. CD44 mRNA is up-regulated in diffuse gastric
adenocarcinoma (left panel), gastric mixed adenocarcinoma (middle
panel), and gastric intestinal type adenocarcinoma (right panel)
when compared to normal gastric mucosa. Data derived from oncomine
(www.oncomine.org).
[0088] FIG. 43. CD44 mRNA is up-regulated in esophageal
adenocarcinoma when compared to esophagus. Data derived from
oncomine (www.oncomine.org)
[0089] FIG. 44. Higher levels of hyaluronan (HA) is detected in
mouse plasma samples derived from the mice bearing breast cancer
(MMTV-PyVT-634Mul/J and FVB-Tg-MMTV-Erbb2-NK1Mul/J mice) or
implanted with gliomas (MSSM-GBMCSC-1 and G1261 cells) when
compared to control mice that have no tumors. Plasma level of HA is
measured using ELISA and biotinylated hsCD44-Fc fusion
proteins.
[0090] FIG. 45. Western Blot of the expression of v-5 epitope
tagged soluble CD44s. Cos-7 (lanes 1, 2, 3) and 293 cells (lanes 4,
5) were transduced with the retroviruses carrying empty expression
vector (lanes 3) and the sCD44sv5 expression construct (lanes 1, 2,
4, 5). Puromycin-resistant pooled populations of Cos-7 and 293
cells were cultured for 48 hours and serum free cell culture
supernatants were collected and analyzed by Western blots using
anti-v5 mAb (Invitrogen).
[0091] FIG. 46. CD44 exon organization. 20 CD44 exons and 10 CD44
variant exons are shown. CD44s consists of exon 1-5, 16-18, and 20.
TM stands for transmembrane and LCT stands for long cytoplasmic
tail. Exon 19 encodes a short cytoplasmic tail. The
NH.sub.2-terminal common extracellular domain of CD44 (SEQ ID#7) is
encoded by exon 1-5. Most of CD44 isoforms contain exon 1-5, exons
16-18, and exon 20 with or without different variant exons
(v1-v10).
DETAILED DESCRIPTION OF THE INVENTION
[0092] The present invention provides pharmaceutical compositions
and methods for treating, preventing, or diagnosing cancers in a
mammal. The present invention further provides pharmaceutical
compositions and methods for treating or preventing gliomas in a
mammal. The present invention provides pharmaceutical compositions
and methods for treating or preventing of glioblastoma multiforme
and other cancer types in a mammal. The present invention is
further directed to pharmaceutical compositions and methods for
sensitizing glioma cells and other types of cancer cells to
oxidative, cytotoxic, and targeted therapeutic stresses for the
treatment of gliomas and other cancer types. Oxidative stresses can
be induced by, but not limited to, chemotherapy or radiation
therapy. In one aspect, CD44 fusion proteins, acting as CD44
antagonists, are administered to a mammal for the treatment,
prevention, or diagnosis of a glioma or other cancer types
including colon cancer, breast cancer, prostate cancer, ovarian
cancer, lung cancer, melanoma, renal cell carcinoma, gastric
cancer, esophageal cancer, pancreatic cancer, liver cancer, and
head-neck cancer. In another aspect, CD44 fusion proteins are
administered alone and/or in combination with other therapeutic
interventions to eliminate and/or suppress cancer stem cells.
Targeted therapies can be inhibitors of EGFR, erbB-2, erbB-3,
erbB-4 and c-Met receptor kinases or other receptor tyrosine
kinases. In another aspect, targeted therapies are inhibitors of
IAPs including cIAPs, XIAP, and survivin. In yet another aspect,
targeted therapies are enhancers/stimulators/stabilizer of p53,
p21, puma, and p38/JNK kinases. In another aspect, targeted
therapies are the agents that promote or induce apoptotic stresses
to cancer cells including inhibitors of PI3K, mTOR, proteasome
inhibitor, and angiogenesis inhibitors. In yet another aspect,
targeted therapies are inhibitors of Wnt signaling pathway.
Gliomas
[0093] Gliomas are the most common type of primary brain cancer and
constitute a spectrum of tumors of variable degrees of
differentiation and malignancy that may arise from the
transformation of neural progenitor cells (Giese et al., 2003;
Maher et al., 2001). The most aggressive of these tumors is grade
IV astrocytoma, also known as glioblastoma multiforme (GBM), that
by virtue of its resistance to chemotherapy, radiotherapy, and
established targeted therapies, is incurable (Davis et al., 1998).
As demonstrated in the present Examples, gliomas express elevated
levels of a major cell surface HA receptor, CD44.
[0094] Resistance to cytotoxic agents, radiation, and targeted
therapies constitutes the major obstacle to successful treatment of
GBM and other malignant cancers. Increasing evidence suggests the
existence of cancer stem cells (CSC), including glioma CSCs, that
are highly resistant to chemo- and radiation therapy and are likely
to be responsible for the recurrence of malignant cancer, including
GBM, following therapeutic intervention (Hambardzumyan et al.,
2008; Reya et al., 2001). Although the implication of CD44 in the
formation and maintenance glioblastoma CSC is just started to be
uncovered, CD44 has been established as a major cell surface CSC
marker in numerous tumors including leukemia and cancers of the
breast, colon, ovary, prostate, pancreas, and head-neck (Croker and
Allan, 2008; Reya et al. 2001; Stamenkovic and Yu, 2009). CD44 has
been shown to be required for engraftment of leukemia CSC in the
bone marrow (Jin et al., 2006; Krause et al., 2006) and to be
functionally relevant for colorectal cancer CSC (Du et al., 2008).
These observations suggest a potentially important role of CD44 in
CSC maintenance and/or function. The present Examples demonstrate
that CD44 attenuates the activation of the Hippo stress/apoptotic
signaling pathway in GBM cells and protects GBM cells from
temozolomide (TMZ) and oxidative stress in vitro and provides a
chemoprotective function in vivo. Furthermore, knockdown of CD44
expression inhibits self-renewal capacity of glioma spheres and
expression of CD44 antagonism, hsCD44s-Fc fusion protein inhibits
in vivo growth of GBMCSCs, suggesting an important role of CD44 in
cancer stem cell maintenance.
Breast Cancer
[0095] Breast cancer is the most common cancer among women in the
United States and the second leading cause of cancer related death
in women. Due to improved early detection and treatment, breast
cancer death rates are going down. However, there are still
estimated 40,170 breast cancer related deaths in year 2009
(http://www.cancer.gov/cancertopics/types/breast), which is largely
caused by the abilities of breast cancer cells to metastasize and
develop resistance to current therapies. This reality urgently begs
for more effective and targeted novel therapies that battle these
deadly abilities of malignant breast cancer. Recent advances in
cancer stem cell (CSC) field have indicated that therapeutic
resistance and recurrence of malignant cancers including breast
cancer are likely due to existence of a small subset of CSCs
including breast CSCs (BCSCs) that are highly resistant to
therapeutic interventions (Al-Hajj et al., 2003; Dean et al., 2005;
Reya et al., 2001). CSCs are characterized by their ability to
self-renew, differentiate into various lineages, and reconstitute
the cellular hierarchy of the tumor (Al-Hajj et al., 2003; Reya et
al., 2001).
[0096] Breast cancers consist of heterogeneous cell populations
including tumor cells and host stroma. Much of cancer research has
been focus on cancer cells. Increasing evidence has indicated that
the host micro-environment plays essential roles in breast cancer
progression and regulating their response to therapies (Al-Hajj et
al., 2003; Liu et al., 2007). Furthermore, maintenance of BCSCs
requires adequate host microenvironment niche. Therefore, it is
essential to develop new therapeutic agents that target BCSCs and
their microenvironment niche in order to eradicate this deadly
disease. Physical interactions and functional cross-talk between
tumor cells and their micro-environment are mediated primarily by
cell surface receptors that are responsible for the cell-cell and
cell-ECM (extracellular matrix) adhesion. CD44 is a major cell
surface receptor for hyaluronan (HA), an abundant component of ECM,
as well as a key marker for CSCs including BCSCs (Collins et al.,
2005; Patrawala et al., 2006; Ponti et al., 2005; Reya et al.,
2001). CD44+/CD24- BCSCs display increased tumorigenicity,
metastatic potential, and chemoresistance (Collins et al., 2005;
Reim et al., 2009; Shipitsin et al., 2007). Accumulation of the
CD44 ligand, HA, in breast cancer stroma is correlated with an
unfavorable prognosis (Tammi et al., 2008).
Prostate Cancer
[0097] Prostate cancer is the second leading cause of
cancer-related death in American men. Prognosis for
hormone-independent/refractory metastatic prostate cancer (HRPC) is
very poor and treatment options for the late stage disease are
limited. Therefore, there is an urgent need to develop more
effective and targeted novel therapies to combat this deadly
disease. To achieve that, it is essential to first identify novel
targets that play key roles in prostate cancer progression,
metastasis, and resistance to chemotherapy. Recent advances in CSC
research demonstrated that CSCs are highly resistant to chemo- and
radio-therapy and are believed to be responsible for tumor
recurrence following therapeutic intervention (Dean et al., 2005;
Reya et al., 2003). CD44 is a predominant cell surface marker for a
variety of human cancer stem or initiating cells including that of
prostate cancers (Collins et al., 2005; Hurt et al., 2008; Maitland
and Collins, 2008).
[0098] Current anti-cancer therapeutic strategies and target
selection are heavily concentrated on frequently mutated kinases
whose activity cancer cells appear to become addicted (Sharma et
al., 2007). Although these approaches are conceptually sound and
supported by notable successes, they are hampered by the emergence
of resistant tumor cells capable of bypassing the targeted
signaling pathways through mechanisms that may be related to the
mutated nature of the target itself. It is now well accepted that
therapeutic interventions targeting only a single signaling
pathway, no matter how seemingly important, are relatively easily
evaded by cancer cells as they acquire new genetic and epigenetic
alterations. An alternative strategy may therefore be to identify
versatile molecules that, unlike the key drivers of oncogenesis,
are not central to any single functional tumor cell property but
participate in multiple functions, including the modulation of
diverse signaling pathways as co-receptors, interactions between
tumor cells and the host tissue microenvironment, and responses of
tumor cells to various forms of stresses. Based on their obvious
usefulness for tumor growth and progression, such molecules are
likely to be upregulated in malignant tumors but unlikely to be
frequently mutated. Selective inhibition of these types of
broad-spectrum targets that play essential roles in mediating
tumor-host interaction and in modulating activities of several
important signaling pathways, especially in combination with chemo-
and radiation therapy, and targeted therapies against these
essential signaling pathways and/or promote/induce stresses to
cancer cells, may therefore overcome the drug resistance obstacle
of current cancer treatment and achieve more efficacious and/or
longer lasting clinical benefits. The present invention indicates
that CD44 is one such target for multiple cancers, and antagonists
of CD44, which includes soluble human CD44 fusion proteins such as
CD44-Fc fusion proteins, are effective anti-cancer agents. Our
preclinical results provide strong support for the therapeutic
potential of targeting CD44 in malignant glioma, breast cancer,
prostate cancer, melanoma, pancreatic cancer, liver cancer, and
head-neck cancer, colon cancer, ovarian cancer, and lung cancer.
For example, FIGS. 3-4, 23, 26-27, 30-31, AND 33 show that shRNAs
against human CD44 inhibit in vivo growth of human glioblastoma (Xu
et al., 2010), colon cancer, breast cancer, prostate cancer, lung
cancer, and ovarian cancer in animal models. FIGS. 12, 17, 36, 40,
and 41 show that expression CD44-Fc fusion proteins inhibits in
vivo growth of human glioblastoma, melanoma, head-neck carcinoma,
pancreatic cancer, and liver cancer in animal models. FIGS. 13, 24,
and 29 show that purified CD44-Fc fusion proteins inhibits in vivo
growth of human glioblastoma, breast cancer, and prostate cancer in
animal models. Together, these comprehensive data establish that
CD44 is a prime therapy target in these cancer types and that CD44
antagonists, including CD44 fusion proteins and shRNAs against
CD44, are effective agents against human glioblastoma, colon
cancer, breast cancer, prostate cancer, lung cancer, ovarian
cancer, melanoma, head-neck carcinoma, pancreatic cancer, and liver
cancer.
[0099] Based on the fact that CD44 is up-regulated and/or plays an
important role in various cancer types, CD44 may serve as
therapeutic target for the following cancers in addition to human
gliomas, colon cancer, breast cancer, prostate cancer, lung cancer,
ovarian cancer, melanoma (Stamenkovic and Yu, 2009), head-neck
carcinoma (Aillles and Prince, 2009; Nelson and Grandis, 2007),
pancreatic cancer (Hong et al., 2009; Klingbeil et al., 2009; Lee
et al., 2008), and liver cancer (Barbour et al., 2003; Yang et al.,
2008), malignant mesothelioma (Ramos-Nino et al., 2007; Tajima et
al., 2010), sarcomas (Yoshida et al., 2008), renal-cell carcinoma
(FIG. 37-38,) (Lim et al., 2008; Lucin et al., 2004; Yildiz et al.,
2004), cancer of the esophagus (FIG. 43) (Li et al., 2005; Nozoe et
al., 2004), Wilms' tumor (Ghanem et al., 2002), bladder carcinoma
(Stavropoulos et al., 2001), multiple myeloma (Mitsiades, 2005;
Ohwada et al., 2008), Gastric Cancer (FIG. 42), and schwannomas
(Bai et al., 2007).
CD44 and Cell Signaling
[0100] CD44 has been implicated in the modulation of several
signaling pathways. It serves as a co-receptor of c-Met (Matzke et
al., 2007) and modulates signals from the ErbB family of RTKs
(Turley et al., 2002). CD44 activates c-Src and focal adhesion
kinase (FAK) (Turley et al., 2002) and promotes cell motility
through activation of Rac1 (Murai et al., 2004). However, no single
core intact pathway that mediates CD44 derived signal has been
established thus far. CD44 interacts with the ERM family proteins
(Tsukita and Yonemura, 1997) and merlin (Morrison et al., 2001;
Sainio et al., 1997), the product of the neurofibromatosis type 2
(NF2) gene. Merlin mutations or loss of merlin expression cause NF2
disease, characterized by the development of schwannomas,
meningiomas, and ependymomas (Gutmann et al., 1997; Kluwe et al.,
1996). In Drosophila, merlin functions upstream of the Hippo
signaling pathway, but a definitive link between merlin and the
mammalian Hippo pathway orthologs has not been fully established.
We have shown recently that merlin is a potent inhibitor of human
GBM growth and that it functions upstream of MST1/2 by activating
MST1/2-Lats2 signaling in glioma cells (Lau et al., 2008). These
observations suggest that the mammalian Hippo signaling pathway may
play an important role in GBM progression. The present invention
demonstrates that cancer cells with depleted endogenous CD44
responded to oxidative and cytotoxic stresses with robust and
sustained phosphorylation/activation of MST1/2 and Lats1/2,
phosphorylation/inactivation of YAP, and reduced expression of
cIAP1/2. These effects correlate with reduced
phosphorylation/inactivation of merlin and increased levels of
cleaved caspase-3. By contrast, a higher level of endogenous CD44
promotes phosphorylation/inactivation of merlin, inhibits the
stress induced activation of the mammalian equivalent of Hippo
signaling pathway, and up-regulates cIAP1/2, leading to the
inhibition of caspase-3 cleavage which is an indicator of
apoptosis. Together, these results place CD44 upstream of the
mammalian Hippo signaling pathway
(merlin-MST1/2-Lats1/2-YAP-cIAP1/2) and suggest a functional role
for CD44 in attenuating tumor cell responses to stress and
stress-induced apoptosis.
[0101] Furthermore, the present invention demonstrates that
knockdown of CD44 results in elevated and sustained activation of
p38/JNK stress kinases, known effectors of MST1/2 kinases, in
glioma cells exposed to oxidative and cytotoxic stress. In
addition, oxidative stress induced a sustained up-regulation of
p53, a known downstream effector of JNK/p38, and its target genes
p21 and puma in CD44-deficient glioma cells, whereas the GBM cells
with high levels of endogenous CD44 attenuated activation of
JNK/p38, and inhibited induction of p53, p21, and puma. These
mechanistic results suggest that CD44 antagonists, including CD44
fusion proteins, can be used in synergy with pharmacological
enhancers/stimulators/stabilizers of p53, p21, puma, and p38/JNK
kinases and with inhibitors of IAPs, including cIAPs and XIAP, to
achieve a better clinical outcome.
[0102] Receptor tyrosine kinases (RTKs) play a central role in a
variety of normal cellular functions, transformation, and tumor
progression. Hepatocyte growth factor (HGF) and its receptor c-Met
are known to promote brain tumor growth and progression (Abounader
and Laterra, 2005). Increased expression of HGF and c-Met
frequently correlates with glioma grade, blood vessel density, and
poor prognosis. Moreover, over expression of HGF and/or c-Met
enhances whereas their inhibition blocks gliomagenesis (Abounader
and Laterra, 2005). In addition, amplification of the EGFR gene
occurs in approximately 40% of GBM cases and constitutes as a
predictor of poor prognosis (Voelzke et al., 2008). The present
invention demonstrates that depletion of CD44 inhibits Erk1/2
activation induced by EGFR ligands and HGF but not by NGF or fetal
bovine serum (FBS; FIG. 7), suggesting that CD44 serves as a
co-receptor/stimulator for these RTKs and enhances their signaling
activity in malignant glioma cells and other cancer types. Although
the precise mechanism whereby CD44 regulates RTK signaling requires
further investigation, its function as an HA receptor provides a
possible explanation. CD44 forms large aggregates on the cell
surface upon engagement by its multivalent ligand, HA. These
aggregates often reside in lipid rafts or other specialized
membrane domains where initiation of multiple signaling events
occurs. In addition, CD44 can be expressed as a cell surface
proteoglycan that binds numerous heparin binding growth factors
including HB-EGF and basic FGF. As an RTK co-receptor, CD44 can
therefore enhance signaling by facilitating RTK oligomerization
and/or presenting the appropriate ligands to the corresponding
RTKs. These mechanistic results suggest that CD44 antagonists,
including CD44 fusion proteins, can be used in synergy with the
pharmacological inhibitors of EGFR, erbB-2, erbB-4 and c-Met
receptor kinases to achieve a better clinical outcome.
CD44 Fusion Proteins
[0103] Because CD44 is a receptor for multiple ligands, the
strategy of using fusion proteins of the extracellular domain of
CD44 with non-CD44 molecules, such as the constant region of human
IgG1 (Fc), is superior to the functional blocking antibodies
against CD44. Each blocking antibody of CD44 can only block the
interaction of one or a few ligands, whereas CD44-Fc fusion
proteins block all the interaction between CD44 and its ligands
mediated by the extracellular domain of CD44. In addition, CD44 is
shed from the cell surface by proteases, which is thought to be a
functionally important process that triggers signaling pathways and
regulates CD44-mediated functions (Stamenkovic and Yu, 2009).
Soluble CD44 fusion proteins contain the domain that interacts with
CD44 sheddase(s); therefore CD44 fusion proteins are capable of
blocking shedding as well as sequestering all the CD44 ligands.
These characteristics of CD44 fusion proteins provide advantages
for antagonizing CD44 function.
[0104] CD44-Fc proteins, which are fusion proteins between the
different segments of the extracellular domain of CD44 with the
constant region of human IgG1 (Fc), act as "trap" type fusion
proteins of a multifunctional transmembrane receptor, which not
only target bulk of tumors, cancer stem cells, but also tumor
microenvironment (such as infiltrating host cells including but not
limited to endothelial cells, pericytes, leukocytes, inflammatory
cells, and fibroblasts, tumor-host cell interaction, and tumor-host
ECM interaction). Key interactions and cross-talk between tumor
cells and their microenvironment are mediated by surface receptors
including cell-cell adhesion and ECM receptors, which provide
potentially attractive therapeutic targets (Marastoni et al.,
2008). The expression of CD44 is often higher in tumor cells,
cancer stem cells, and tumor microenvironment, but lower in normal
tissues; therefore, CD44 serves as an ideal target for cancer
therapy.
[0105] CD44 protein consists of an extracellular domain with an
NH.sub.2-terminal HA-binding region and a membrane-proximal region,
a transmembrane domain (TM), and a COOH-terminal cytoplasmic tail
(CT) (Peach et al., 1993; Stamenkovic et al., 1989). There is a
single CD44 gene containing 20 exons. At least 10 of these exons,
exons 6-15 or variant exons v1-v10, can be alternatively spliced to
give rise to numerous CD44 variants (Screaton et al., 1993;
Screaton et al., 1992). The standard form of CD44 (CD44s) is a
product of alternative splicing of transcript and consists of all
the common exons 1-5, 16-18 and 20.
[0106] In one embodiment of the present invention, CD44 fusion
proteins, acting as CD44 antagonists, are administered to a mammal
for the treatment or prevention of a glioma or other cancer types.
In one aspect, CD44 fusion proteins are administered prior to,
simultaneously with, or after an additional anti-cancer therapy or
surgical removal of tumors including glioma. CD44 is important for
cancer stem cells as we have shown that CD44 depletion inhibits the
formation of glioma spheres (FIG. 18), mammospheres (FIG. 28C), and
ovarian CSC spheres (FIG. 34E) and that overexpression of CD44
inhibits GBMCSC growth in vivo (FIG. 17D) and purified CD44-Fc
fusion proteins inhibited growth of BCSCs in vivo (FIG. 29).
Because CSCs often survive cancer therapies, treatment with CD44
fusion proteins following these therapies is important to prevent
the recurrence and metastasis of cancer, even in the absence of
visible disease. In another aspect, the CD44 fusion proteins are
administered prior to, simultaneously with, or after a treatment
which causes cytotoxic stresses or other forms of stresses. In yet
another aspect, CD44 fusion proteins are administered as single
agents or along with other anti-cancer therapies prior to or after
surgery to treat glioma and other cancer types, wherein the
administration of CD44 fusion proteins and the additional
therapeutic agents provides a synergistic effect. In one aspect,
CD44 fusion protein is administrated as purified protein. In
another aspect, CD44 fusion protein is administrated in the form of
viral expression vector with or without being packaged into viral
particles or using nanoparticles as carriers. In one aspect, the
viral particle is retrovirus, lentivirus, adenovirus, or
adeno-associated virus (AAV). In one aspect, the adenovirus is a
replication-impaired, non-integrating, serotype 2, 5, 6, 7, or 8
adenoviral vector. In one aspect, the CD44 fusion protein is a
CD44-Fc fusion protein, which comprises the constant region of
human IgG1 fused to different segments of the extracellular domain
of CD44. In another aspect, the CD44 extracellular domain is
derived from CD44s, CD44v3-v10, CD44v6-v10, CD44v8-v10, CD44sR41A,
CD44v3-v10R41A, CD44v6-v10R41A, and CD44v8-v10R41A. In yet another
aspect, the CD44 extracellular domain is derived from CD44v4-v10,
CD44v5-v10, CD44v7-v10, CD44v9-v10, CD44v3, CD44v4, CD44v5, CD44v6,
CD44v7, CD44v9, CD44v10, or the above CD44 isoforms containing the
R41A mutation. In yet another aspect, the CD44 extracellular domain
is derived from different combinations of exons 1-17, different
deletions, mutations, duplication, or multiplication of the
different segments of the extracellular domain of CD44.
[0107] The extracellular domain of CD44 is encoded by exon 1-5,
v1-v10 (or exon 6-10), exon 16, and exon 17. The extracellular
domain of CD44s consists of exon 1-5, 16, and 17. CD44 variants
(CD44v2-v10, CD44v3-v10, CD44v8-v10, CD44v4-v10, CD44v5-v10,
CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9, CD44v8,
CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, and CD44v2) consist of exon
1-5, different combinations of the variant exons (v2-v10), exon 16,
and exon 17 (FIG. 46).
TABLE-US-00001 TABLE 1 Nucleotide and Amino Acid Sequences for the
constant region (Fc) of Homo sapien Immunoglobulin Heavy Constant
Gamma 1, Human CD44 Wild Type Extracellular Domains, Human CD44
Extracellular Domains containing R41A mutation, and CD44-Fc Fusion
Proteins SEQ ID Protein Sequence No. Constant gacaaaactc 1 region
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc ttectettcc
(Fc) of Homo ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
tgcgtggtgg sapien tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac ggcgtggagg immuno- tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac cgtgtggtca globulin gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag tgcaaggtct heavy ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc constant
gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca
gamma 1 gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
ctctccctgt ctccgggtaa atga DKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH 41 EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ
QGNVFSCSVM HEALHNHYTQ KSLSLSPGK Human CD44 atggac aagttttggt
ggcacgcagc ctggggactc tgcctcgtgc cgctgagcct ggcg 2 signal peptide
MDKFWWHAAWGLCLVPLSLA 42 Constant atggac aagttttggt ggcacgcagc
ctggggactc tgcctcgtgc cgctgagcct ggcg gacaaaactc 3 region(Fc) of
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc ttcctcttcc
immunoglobulin ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca tgcgtggtgg heavy constant tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac ggcgtggagg gamma 1 with tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca CD44 signal
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct
peptide ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
ctctccctgt ctccgggtaa atga MDKFWWHAAWGLCLVPLSLA DKTHTCPP CPAPELLGGP
SVFLFPPKPK 43 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK
TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS
KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK Wild type atggac
aagttttggt ggcacgcagc ctggggactc tgcctcgtgc 4 extracellular
cgctgagcct ggcg cagatc gatttgaata taacctgccg ctttgcaggt gtattccacg
domain of tggagaaaaa tggtcgctac agcatctctc ggacggaggc cgctgacctc
tgcaaggctt CD44s tcaatagcac cttgcccaca atggcccaga tggagaaagc
tctgagcatc ggatttgaga (exon 1-5, 16 cctgcaggta tgggttcata
gaagggcacg tggtgattcc ccggatccac cccaactcca and17) tctgtgcagc
aaacaacaca g,gggtgtaca tcctcacatc caacacctcc cagtatgaca catattgctt
caatgcttca gctccacctg aagaagattg tacatcagtc acagacctgc ccaatgcctt
tgatggacca attaccataa ctattgttaa ccgtgatggc acccgctatg tccagaaagg
agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg atgacgtgag
cagcggctcc tccagtgaaa ggagcagcac ncaggaggt tacatctta acaccattc
tactgtacac cccatcccag acgaagacag tccctggatc accgacagca cagacagaat
ccctgctacc aga gac caagacacat tccaccccag tggggggtcc cataccactc
atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca aacacaacct
ctggtcctat aaggacaccc caaattccag aa
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI 44
SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSI CAANNT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPAT R
DQDTF HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE extracellular atggac
aagttttggt ggcacgcagc ctggggactc tgcctcgtgc 5 domain of CD44s
cgctgagcct ggcg cagatc gatttgaata taacctgccg ctttgcaggt gtattccacg
with a R41A tggagaaaaa t ggt gcc tac agc atc tctc ggacggaggc
cgctgacctc tgcaaggctt mutation (exon tcaatagcac cttgcccaca
atggcccaga tggagaaagc tctgagcatc ggatttgaga 1-5, 16, and 17)
cctgcaggta tgggttcata gaagggcacg tggtgattcc ccggatccac cccaactcca
tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc caacacctcc cagtatgaca
catattgctt caatgcttca gctccacctg aagaagattg tacatcagtc acagacctgc
ccaatgcctt tgatggacca attaccataa ctattgttaa ccgtgatggc acccgetatg
tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg
atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt tacatctttt
acaccttttc tactgtacac cccatcccag acgaagacag tccctggatc accgacagca
cagacagaat ccctgctacc aga gac caagacacat tccaccccag tggggggtcc
cataccactc atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca
aacacaacet ctggtcetat aaggacaccc caaattccag aa
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGAYSI 45
SRTEAADLCKAENSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSI CAANNT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPAT R
DQDTF HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE CD44 NH.sub.2-
atggac aagttttggt ggcacgcagc ctggggactc tgcctcgtgc 6 terminal
cgctgagcct ggcg cagatc gatttgaata taacctgccg ctttgcaggt gtattccacg
common tggagaaaaa t ggt gcc tac agc atc tct c gg acggaggc
cgctgacctc tgcaaggctt extracellular tcaatagcac cttgcccaca
atggcccaga tggagaaagc tctgagcatc ggatttgaga domain with a
cctgcaggta tgggttcata gaagggcacg tggtgattcc ccggatccac cccaactcca
R41A mutation tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc
caacacctcc cagtatgaca (exon 1-5) catattgctt caatgcttca gctccacctg
aagaagattg tacatcagtc acagacctgc ccaatgcctt tgatggacca attaccataa
ctattgttaa ccgtgatggc acccgctatg tccagaaagg agaatacaga acgaatcctg
aagacatcta ccccagcaac cctactgatg atgacgtgag cagcggctcc tccagtgaaa
ggagcagcac ttcaggaggt tacatctttt acaccttttc tactgtacac cccatcccag
acgaagacag tccctggatc accgacagca cagacagaat ccctgctacc agt
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGAYSI 46
SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAAN NT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPATS
Wild type atggac aagttttggt ggcacgcagc ctggggactc tgcctcgtgc 7 CD44
NH.sub.2- cgctgagcct ggcg cagatc gatttgaata taacctgccg ctttgcaggt
gtattccacg terminal tggagaaaaa tggtcgctac agcatctctc ggacggaggc
cgctgacctc tgcaaggctt common tcaatagcac cttgcccaca atggcccaga
tggagaaagc tctgagcatc ggatttgaga extracellular cctgcaggta
tgggttcata gaagggcacg tggtgattcc ccggatccac cccaactcca domain
tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc caacacctcc cagtatgaca
(exon 1-5) catattgctt caatgcttca gctccacctg aagaagattg tacatcagtc
acagacctgc ccaatgcctt tgatggacca attaccataa ctattgttaa ccgtgatggc
acccgctatg tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac
cctactgatg atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt
tacatctat acaccttttc tactgtacac cccatcccag acgaagacag tccctggatc
accgacagca cagacagaat ccctgctacc agt
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI 47
SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAAN NT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPATS
Partial acgtctt caaataccat ctcagcaggc tgggagccaa CD44v3-v10
atgaagaaaa tgaagatgaa agagacagac acctcagttt ttctggatca ggcattgatg 8
extracellular atgatgaaga ttttatctcc agcacc attt caaccacacc
acgggctttt gaccacacaa domain aacagaacca ggactggacc cagtggaacc
caagccattc aaatccggaa gtgctacttc (a part of the agacaaccac
aaggatgact gatgtagaca gaaatggcac cactgcttat gaaggaaact
extracellular ggaacccaga agcacaccct cccctcattc accatgagca
tcatgaggaa gaagagaccc domain cacattctac aagcacaatc caggcaactc
ctagtagtac aacggaagaa acagctaccc containing agaaggaaca gtggtttggc
aacagatggc atgagggata tcgccaaaca cccaaagaag exons v3-v10,
actcccattc gacaacaggg acagctgcag cctcagctca taccagccat ccaatgcaag
16,and 17) gaaggacaac accaagccca gaggacagtt cctggactga tttcttcaac
ccaatctcac accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc
agtcatagta taacgcttca gcctactgca aatccaaaca caggtttggt ggaagatttg
gacaggacag gacctctttc aatgacaacg cagcagagta attctcagag cttctctaca
tcacatgaag gcttggaaga agataaagac catccaacaa cttctactct gacatcaagc
aataggaatg atgtcacagg tggaagaaga gacccaaatc attctgaagg ctcaactact
ttactggaag gttatacctc tcattaccca cacacgaagg aaagcaggac cttcatccca
gtgacctcag ctaagactgg gtcctttgga gttactgcag ttactgttgg agattccaac
tctaatgtca atcgttcctt atca ggagac caagacacat tccaccccag tggggggtcc
cataccactc atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca
aacacaacct ctggtcctat aaggacaccc caaattccag aa
TSSNTISAGWEPNEENEDERDRHLSFSGSGIDDDEDFISSTISTTPRAFDHTK 48
QNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNGTTAYEGNWNPEAHPPLIHHE HHEEEE
TPHSTSTIQATPSSTTEETATQICEQWFGNRWHEGYRQTPICEDSHSTTGTAAASA HTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRMDMDSSHSITLQPTANPNT GLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPN HSE
GSTILLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial ga tatggactcc
agtcatagta 9 CD44v8-v10 extracellular taacgcttca gcctactgca
aatccaaaca caggtttggt ggaagatttg gacaggacag domain gacctctttc
aatgacaacg cagcagagta attctcagag cttctctaca tcacatgaag (a part of
the gcttggaaga agataaagac catccaacaa cttctactct gacatcaagc
aataggaatg extracellular atgtcacagg tggaagaaga gacccaaatc
attctgaagg ctcaactact ttactggaag domain gttatacctc tcattaccca
cacacgaagg aaagcaggac cttcatccca gtgacctcag containing ctaagactgg
gtcctttgga gttactgcag ttactgttgg agattccaac tctaatgtca exon v8-v10,
atcgttcctt atca ggagac caagacacat tccaccccag tggggggtcc cataccactc
16 and 17) atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca
aacacaacct ctggtcctat aaggacaccc caaattccag aa
DMDSSHSITLQPTANPNTGLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPITSTLTSSNRNDVTGGRRDPN 49 HSE
GSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE CD44s-Fc atggac aagttttggt
ggcacgcagc ctggggactc tgcctcgtgc 10 (exon 1-5, 16, cgctgagcct ggcg
cagatc gatttgaata taacctgccg ctttgcaggt gtattccacg and 17 of
tggagaaaaa tggtcgctac agcatctctc ggacggaggc cgctgacctc tgcaaggctt
CD44- tcaatagcac cttgcccaca atggcccaga tggagaaagc tctgagcatc
ggatttgaga CAATTG cctgcaggta tgggttcata gaagggcacg tggtgattcc
ccggatccac cccaactcca -Fc) tctgtgcagc aaacaacaca ggggtgtaca
tcctcacatc caacacctcc cagtatgaca
catattgctt caatgcttca gctccacctg aagaagattg tacatcagtc acagacctgc
ccaatgcctt tgatggacca attaccataa ctattgttaa ccgtgatggc acccgctatg
tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg
atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt tacatctttt
acaccttttc tactgtacac cccatcccag acgaagacag tccctggatc accgacagca
cagacagaat ccctgctacc aga gac caagacacat tccaccccag tggggggtcc
cataccactc atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca
aacacaacct ctggtcctat aaggacaccc caaattccag aa CAATTG gacaaaactc
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct
ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt
ctccgggtaa atga MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI 50
SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAAN NT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD RIPAT R
DQDTF HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE QL DKTHTCPP
CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK
TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS
KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK CD44v3-v10-Fc atggac
aagttttggt ggcacgcagc ctggggactc tgcctcgtgc (exon 1-5, cgctgagcct
ggcgcagatc gatttgaata taacctgccg ctttgcaggt gtattccacg tggagaaaaa
tggtcgctac 11 v3-v10, 16, agcatctctc ggacggaggc cgctgacctc
tgcaaggctt tcaatagcac cttgcccaca atggcccaga and 17 of tggagaaagc
tctgagcatc ggatttgagacctgcaggta tgggttcata gaagggcacg tggtgattcc
ccggatccac CD44-CAATTG- cccaactcca tctgtgcagc aaacaacaca ggggtgtaca
tcctcacatc caacacctcc cagtatgaca Fc) catattgctt caatgatca
gctccacctg aagaagattg tacatcagtc acagacctgc ccaatgcctt tgatggacca
CD44v3-v10-Fc attaccataa ctattgttaa ccgtgatggc acccgctatg
tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg
atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt tacatctttt
acaccttttc tactgtacac cccatcccag acgaagacag tccctggatc accgacagca
cagacagaat ccctgctacc agtacgtctt caaataccat ctcagcaggc tgggagccaa
atgaagaaaa tgaagatgaa agagacagac acctcagttt ttctggatca ggcattgatg
atgatgaaga ttttatctcc agcaccattt caaccacacc acgggctttt gaccacacaa
aacagaacca ggactggacc cagtggaacc caagccattc aaatccggaa gtgctacttc
agacaaccac aaggatgact gatgtagaca gaaatggcac cactgcttat gaaggaaact
ggaacccaga agcacaccct cccctcattc accatgagca tcatgaggaa gaagagaccc
cacattctac aagcacaatc caggcaactc ctagtagtac aacggaagaa acagctaccc
agaaggaaca gtggtttggc aacagatggc atgagggata tcgccaaaca cccaaagaag
actcccattc gacaacaggg acagctgcag cctcagctca taccagccat ccaatgcaag
gaaggacaac accaagccca gaggacagtt cctggactga tttcttcaac ccaatctcac
accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc agtcatagta
taacgcttca gcctactgca aatccaaaca caggtttggt ggaagatttg gacaggacag
gacctctttc aatgacaacg cagcagagta attctcagag cttctctaca tcacatgaag
gcttggaaga agataaagac catccaacaa cttctactct gacatcaagc aataggaatg
atgtcacagg tggaagaaga gacccaaatc attctgaagg ctcaactact ttactggaag
gttatacctc tcattaccca cacacgaagg aaagcaggac cttcatccca gtgacctcag
ctaagactgg gtcctttgga gttactgcag ttactgttgg agattccaac tctaatgtca
atcgttcctt atca ggagac caagacacat tccaccccag tggggggtcc cataccactc
atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca aacacaacct
ctggtcctat aaggacaccc caaattccag aa CAATTG gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta
cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa
atga MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI 51
SRTEAADLCKAFNSTLPTMAQMEICALSIGFETCRYGFIEGHVVIPRIHPNSICAAN NT
GVYILTSNTSQVDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPATS
TSSNTISAGWEPNEENEDERDRHLSFSGSGIDDDEDFISST ISTTPRAFDHTK
QNQDWTQWNPSHSNPEVLLQTTTRMT DVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEE
TPHSTSTI QATPSSTTEETATQKEQWFGNRWHEGYRQTPICEDSHSTTGTAA ASAHTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRM DMDSSHSITLQPTANPNTGLVE
DLDRTGPLSMTT QQSNSQSFSTSHEGLEEDKDHPTTSTLTSS NRNDVTGGRRDPNHSE
GSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLS GDQDTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE QL DKTHTCPP CPAPELLGGP
SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ
QGNVFSCSVM HEALHNHYTQ KSLSLSPGK CD44v8-v10-FC Atg gac aagttttggt
ggcacgcagc ctggggactc tgcctcgtgc 12 (exon 1-5, cgctgagcct ggcg
cagatc gatttgaata taacctgccg ctttgcaggt gtattccacg v8-v10, 16,
tggagaaaaa tggtcgctac agcatctctc ggacggaggc cgctgacctc tgcaaggctt
and 17 of tcaatagcac cttgcccaca atggcccaga tggagaaagc tctgagcatc
ggatttgaga CD44-CAATTG-Fc cctgcaggta tgggttcata gaagggcacg
tggtgattcc ccggatccac cccaactcca CD44v8-v10-FC tctgtgcagc
aaacaacaca ggggtgtaca tcctcacatc caacacctcc cagtatgaca catattgctt
caatgcttca gciccacctg aagaagattg tacatcagtc acagacctgc ccaatgcctt
tgatggacca attaccataa ctattgttaa ccgtgatggc acccgctatg tccagaaagg
agaatacaga acgaaicctg aagacatcta ccccagcaac cctactgatg atgacgtgag
cageggctec tccagtgaaa ggagcagcac ttcaggaggt tacatctttt acaccttttc
tactgtacac cccatcccag acgaagacag tccctggatc accgacagca cagacagaat
ccctgctacc agt ga tatggactcc agtcatagta taaegcttea gcctactgca
aatccaaaca caggittggt ggaagatttg gacaggacag gacctcmc aatgacaacg
cagcagagia aitctcagag cttctciaca icacatgaag gcriggaaga agataaagac
catccaacaa cttctactct gacatcaagc aafaggaatg atgtcacagg tggaagaaga
gacccaaatc attctgaagg ctcaactact ttactggaag gttatacctc tcattaccca
cacacgaagg aaagcaggac cttcatccca gtgacctcag ctaagactgg gtcctttgga
gttactgcag ttactgttgg agattccaac tctaatgtca ategttcett atcaggagac
caagacacat tccaccccag tggggggtcc cataccactc atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct ctggtcctat aaggacaccc
caaattccag aa CAATTG gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca
agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccgegggagg
agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagecaaa gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttcratc
ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc gacggctcct tcttcctcia cagcaagctc accgtggaca
agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgeatgag gctcigcaca
accaciacae gcagaagagc ctctccctgt ctccgggtaa atga
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSI 52 SRTEAADLCKAFNSILR1
MAQMEKAli5IGFETCRYGFIEGHVVIPRIHPNSICAAN NT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD R IPATS
DMDSSHSITLQPTANPNTGLVE DLDRTGPLSMTT
QQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPNMSE
GSTTLLEGYTSHYPIITKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLS GDQDTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE QL DKTHTCPP CPAPELLGGP
SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK IXPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIFKTISK AKGQPREPQV YTLPPSRDEL
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ
QGNVFSCSVM HEALHNHYTQ KSLSLSPGK CD44sR41A-Fc atggac aagttttggt
ggcacgcagc ctggggactc tgcctcgtgc cgctgagcct ggcg cagatc gatttgaata
13 taacctgccg ctttgcaggt gtattccacg tggagaaaaa t ggt gcc tac
agcatctctc ggacggaggc cgctgacctc tgcaaggctt tcaatagcac cttgcccaca
atggcccaga tggagaaagc tctgagcatc ggatttgaga cctgcaggta tgggttcata
gaagggcacg tggtgattcc ccggatccac cccaactcca tctgtgcagc aaacaacaca
ggggtgtaca tcctcacatc caacacctcc cagtatgaca catattgctt caatgcttca
gctccacctg aagaagattg tacatcagtc acagacctgc ccaatgcctt tgatggacca
attaccataa ctattgttaa ccgtgatggc acccgctatg tccagaaagg agaatacaga
acgaatcctg aagacatcta ccccagcaac cctactgatg atgacgtgag cagcggctcc
tccagtgaaa ggagcagcac ttcaggaggt tacatctttt acaccttttc tactgtacac
cccatcccag acgaagacag tccctggatc accgacagca cagacagaat ccctgctacc
aga gac caagacacat tccaccccag tggggggtcc cataccactc atggatctga
atcagatgga cactcacatg ggagtcaaga aggtggagca aacacaacct ctggtcctat
aaggacaccc caaattccag aa CAATTG gacaaaactc acacatgccc accgtgccca
gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga
MDKFWWHAAWGLCLVPLSLAQIDLNITCR_FAGVFHVEKNGAYSI 53
SRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPPdHPNSICAAN NT
GVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQK GE
YRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTD RIPAT R
DQDTF HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE QL DKTHTCPP
CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK
TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS
KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK CD44-Fc with a Wild type
CD44 NH2-terminal common extracellular domain (SEQ ID No. 7, exon
1-5) 14 MfeI +/- various CD44 extracellular domains (SEQ ID 8, 9,
or 18-30) + CAATTG (GlnLeu) + restriction SEQID NO. 1 enzyme
cutting site CD44-Fc without Wild type CD44 NH2-terminal common
extracellular domain (SEQ ID No. 7) 15 a MfeI CD44 extracellular
domain (SEQ ID 8, 9, or 18-30) + SEQID NO. 1 restriction enzyme
cutting site CD44R41A-Fc CD44 NH2-terminal common extracellular
domain with R41A mutation (SEQ ID +1906) +/- 16 with a MfeI various
CD44 extracellular domain (SEQ ID 8, 9, or 18- 30) + CAATTG
(GlnLeu) +
restriction SEQID No. 1 enxyme cutting site CD44R41A-Fc CD44
NH2-terminal common extracellular domain with R41A mutation (SEQ ID
NO 17 without 6). restriction enzyme cutting site Partial attt
caaccacacc acgggctttt gaccacacaa 18 CD44v4-v10 aacagaacca
ggactggacc cagtggaacc caagccattc aaatccggaa gtgctacttc extrcellular
agacaaccac aaggatgact gatgtagaca gaaatggcac cactgcttat gaaggaaact
domain ggaacccaga agcacaccct cccctcattc accatgagca tcatgaggaa
gaagagaccc (a part cacattctac aagcacaatc caggcaactc ctagtagtac
aacggaagaa acagctaccc of the agaaggaaca gtggtUggc aacagatggc
atgagggata tcgccaaaca cccaaagaag extracellular actcccattc
gacaacaggg acagctgcag cctcagctca taccagccat ccaatgcaag domain
gaaggacaac accaagccca gaggacagtt cctggactga tttcttcaac ccaatctcac
containing accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc
agtcatagta exon v4-v10,16, taacgcttca gcctactgca aatccaaaca
caggtttggt ggaagatttg gacaggacag and 17) gacctctttc aatgacaacg
cagcagagta attctcagag cttctctaca tcacatgaag gcttggaaga agataaagac
catccaacaa cttctactct gacatcaagc aataggaatg atgtcacagg tggaagaaga
gacccaaatc attctgaagg ctcaactact ttactggaag gttatacctc tcattaccca
cacacgaagg aaagcaggac cttcatccca gtgacctcag ctaagactgg gtcctttgga
gttactgcag ttactgttgg agattccaac tctaatgtca atcgttcctt atca ggagac
caagacacat tccaccccag tggggggtcc cataccactc atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct ctggtcctat aaggacaccc
caaattccag aa ISTTPRAFDHTK 58
QNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNGTTAYEGNWNPEAHPPLIHHE HHEEEE
TPHSTSTIQATPSSTTEETATQICEQWEGNRWHEGYRQTPKEDSHSTTGTAAASA HTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRMDMDSSHSITLQPTANPNT GLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVIGGRRDPN HSE
GSTILLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial CD44v5- gatgtagaca
gaaatggcac cactgcttat gaaggaaact 19 v10 ggaacccaga agcacaccct
cccctcattc accatgagca tcatgaggaa gaagagaccc extracellular
cacattctac aagcacaatc caggcaactc ctagtagtac aacggaagaa acagctaccc
domain agaaggaaca gtggtttggc aacagatggc atgagggata tcgccaaaca
cccaaagaag (a part of the actcccattc gacaacaggg acagctgcag
cctcagctca taccagccat ccaatgcaag extracellular gaaggacaac
accaagccca gaggacagtt cctggactga tttcttcaac ccaatctcac domain
accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc agtcatagta
containing taacgcttca gcctactgca aatccaaaca caggtttggt ggaagatttg
gacaggacag exon v5-v10, gacctctttc aatgacaacg cagcagagta attctcagag
cttctctaca tcacatgaag 16, and 17) gcttggaaga agataaagac catccaacaa
cttctactct gacatcaagc aataggaatg atgtcacagg tggaagaaga gacccaaatc
attctgaagg ctcaactact ttactggaag gttatacctc tcattaccca cacacgaagg
aaagcaggac cttcatccca gtgacctcag ctaagactgg gtcctttgga gttactgcag
ttactgttgg agattccaac tctaatgtca atcgttcctt atca ggagac caagacacat
tccaccccag tggggggtcc cataccactc atggatctga atcagatgga cactcacatg
ggagtcaaga aggtggagca aacacaacct ctggtcctat aaggacaccc caaattccag
aa DVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEE 59
TPHSTSTIQATPSSTTEETATQ10EQWFGNRWHEGYRQTPKEDSHSTTGTAAASA HTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRAMDMDSSHSITLQPTANPNT GLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDICDHPTTSTLTSSNRNDVTGGRRDPN HSE
GSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHITHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial caggcaactc
ctagtagtac aacggaagaa acagctaccc 20 CD44v6-v10 agaaggaaca
gtggtttggc aacagatggc atgagggata tcgccaaaca cccaaagaag
extracellular actcccattc gacaacaggg acagctgcag cctcagctca
taccagccat ccaatgcaag domain (a part gaaggacaac accaagccca
gaggacagtt cctggactga tttcttcaac ccaatctcac of extracellular
accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc agtcatagta
domain taacgcttca gcctactgca aatccaaaca caggtttggt ggaagatttg
gacaggacag containing gacctctttc aatgacaacg cagcagagta attctcagag
cttctctaca tcacatgaag exon v6-v10, gcttggaaga agataaagac catccaacaa
cttctactct gacatcaagc aataggaatg 16, and 17) atgtcacagg tggaagaaga
gacccaaatc attctgaagg ctcaactact ttactggaag gttatacctc tcattaccca
cacacgaagg aaagcaggac cttcatccca gtgacctcag ctaagactgg gtcctttgga
gttactgcag ttactgttgg agattccaac tctaatgtca atcgttcctt atca ggagac
caagacacat tccaccccag tggggggtcc cataccactc atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct ctggtcctat aaggacaccc
caaattccag aa QATPSS ITEETATQKEQWFGNRWHEGYRQTPKEDSHSTTGTAAASAHTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRAMDMDSSHSITLQPTANPNT 60 GLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPN HSE
GSTTLLEGYTSHYPHTICESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial g cctcagctca
taccagccat ccaatgcaag 21 CD44v7-v10 gaaggacaac accaagccca
gaggacagtt cctggactga tttcttcaac ccaatctcac extracellular
accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc agtcatagta
domain taacgcttca gcctactgca aatccaaaca caggtttggt ggaagatttg
gacaggacag (a part of the gacctctttc aatgacaacg cagcagagta
attctcagag cttctctaca tcacatgaag extracellular gcttggaaga
agataaagac catccaacaa cttctactct gacatcaagc aataggaatg domain
atgtcacagg tggaagaaga gacccaaatc attctgaagg ctcaactact ttactggaag
containing gttatacctc tcattaccca cacacgaagg aaagcaggac cttcatccca
gtgacctcag exon v7- ctaagactgg gtcctttgga gttactgcag ttactgttgg
agattccaac tctaatgtca v10, 16, atcgttcctt atca ggagac caagacacat
tccaccccag tggggggtcc cataccactc and 17) atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct ctggtcctat aaggacaccc
caaattccag aa ASAHTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRMDMDSSHSITLQPTANPNT 61 GLVE
DLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPN HSE
GSTTLLEGYTSHYPHTICESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial cagcagagta
attctcagag cttctctaca tcacatgaag 22 CD44v9-v10 gcttggaaga
agataaagac catccaacaa cttctactct gacatcaagc aataggaatg
extracellular atgtcacagg tggaagaaga gacccaaatc attctgaagg
ctcaactact ttactggaag domain gttatacctc tcattaccca cacacgaagg
aaagcaggac cttcatccca gtgacctcag (a part of the ctaagactgg
gtcctttgga gttactgcag ttactgttgg agattccaac tctaatgtca
extracellular atcgttcctt atca ggagac caagacacat tccaccccag
tggggggtcc cataccactc domain atggatctga atcagatgga cactcacatg
ggagtcaaga aggtggagca aacacaacct containing ctggtcctat aaggacaccc
caaattccag aa exon v9-v10,
QQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPNHSE 62 16, and 17)
GSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial CD44v10 aataggaatg
23 extracellular atgtcacagg tggaagaaga gacccaaatc attctgaagg
ctcaactact ttactggaag domain gttatacctc tcattaccca cacacgaagg
aaagcaggac cttcatccca gtgacctcag (a part of ctaagactgg gtcctttgga
gttactgcag ttactgttgg agattccaac tctaatgtca extracellular
atcgttcctt atca ggagac caagacacat tccaccccag tggggggtcc cataccactc
domain atggatctga atcagatgga cactcacatg ggagtcaaga aggtggagca
aacacaacct containing ctggtcctat aaggacaccc caaattccag aa exon v10,
NRNDVTGGRRDPNHSE 63 16, and
GSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQ 17) DTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE Partial CD44v9 cagcagagta
attctcagag cnctctaca tcacatgaag 24 extracellular gcttggaaga
agataaagac catccaacaa cttctactct gacatcaagc domain ggagac
caagacacat tccaccccag tggggggtcc cataccactc (a part of atggatctga
atcagatgga cactcacatg ggagtcaaga aggtggagca aacacaacct
extracellular ctggtcctat aaggacaccc caaattccag aa domain
QQSNSQSFSTSHEGLEEDKDHPTTSTLTS GDQDTF 64 containing
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE exon v9, 16, and 17)
Partial CD44v8 ga tatggactcc agtcatagta 25 extracellular taacgcttca
gcctactgca aatccaaaca caggtttggt ggaagatttg gacaggacag domain
gacctctttc aatgacaacg (a part of ggagac caagacacat tccaccccag
tggggggtcc cataccactc extracellular atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct domain ctggtcctat
aaggacaccc caaattccag aa containing DMDSSHSITLQPTANPNTGLVE
DLDRTGPLSMTT GDQDTF 65 exon v8,
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE 16, and 17) Partial CD44v7
g cctcagctca taccagccat ccaatgcaag gaaggacaac accaagccca gaggacagtt
cctggactga 26 extracellular tttcttcaac ccaatctcac accccatggg
acgaggtcat caagcaggaa gaaggatg domain ggagac caagacacat tccaccccag
tggggggtcc cataccactc (a part of atggatctga atcagatgga cactcacatg
ggagtcaaga aggtggagca aacacaacct extracellular ctggtcctat
aaggacaccc caaattccag aa domain ASAHTSH
PMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRM 66 containing
GDQDTFHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE exon v7, 16, and 17)
Partial CD44v6 caggcaactc ctagtagtac aacggaagaa acagctaccc
agaaggaaca gtggtttggc aacagatggc 27 extracellular atgagggata
tcgccaaaca cccaaagaag actcccattc gacaacaggg acagctgca domain ggagac
caagacacat tccaccccag tggggggtcc cataccactc atggatctga atcagatgga
cactcacatg (a part of ggagtcaaga aggtggagca aacacaacct ctggtcctat
aaggacaccc caaattccag aa extracellular
QATPSSTTEETATQKEQWEGNRWHEGYRQTPKEDSHSTTGTAA 67 domain GDQDTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE containing exon v6, 16,
and 17) Partial CD44v5 gatgtagaca gaaatggcac cactgcttat gaaggaaact
28 extracellular ggaacccaga agcacaccct cccctcattc accatgagca
tcatgaggaa gaagagaccc cacattctac domain aagcacaatc (a part of
ggagac caagacacat tccaccccag tggggggtcc cataccactc atggatctga
atcagatgga cactcacatg extracellular ggagtcaaga aggtggagca
aacacaacct domain ctggtcctat aaggacaccc caaattccag aa containing
DVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEE TPHSTSTI 68 exon v5, GDQDTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE 16, and 17) Partial CD44v4
attt caaccacacc acgggctttt gaccacacaa aacagaacca ggactggacc
cagtggaacc caagccattc 29 extracellular aaatccggaa gtgctacttc
agacaaccac aaggatgact ggagac caagacacat tccaccccag tggggggtcc
domain cataccactc atggatctga atcagatgga cactcacatg ggagtcaaga
aggtggagca aacacaacct ctggtcctat (a part of aaggacaccc caaattccag
aa extracellular ISTTPRAFDHTK QNQDWTQWNPSHSNPEVLLQTTTRMT 69 domain
GDQDTF HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE containing: exon
v4, 16, and 17) Partial CD44v3 acgtctt caaataccat ctcagcaggc
tgggagccaa 30 extracellular atgaagaaaa tgaagatgaa agagacagac
acctcagttt ttctggatca ggcattgatg
domain atgatgaaga ttttatctcc agcacc (a part of ggagac caagacacat
tccaccccag tggggggtcc cataccactc atggatctga atcagatgga cactcacatg
extracellular ggagtcaaga aggtggagca aacacaacct ctggtcctat
aaggacaccc caaattccag aa domain
TSSNTISAGWEPNEENEDERDRHLSFSGSGIDDDEDFISST 70 containing GDQDTF
HPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPE exon v3, 16, and 17)
[0108] A fusion protein (SEQ ID NO: 3) comprising the constant
region (Fc) of immunoglobulin heavy constant gamma 1 (SEQ ID NO: 1)
and the CD44 signal peptide (SEQ ID NO: 2) can be used as a
negative control for the anti-tumor activity of the CD44-Fc fusion
proteins. In some embodiments, the CD44 extracellular domain of the
CD44 fusion protein comprises fragments of CD44 extracellular
domains, which is encoded by different combinations of the exons
1-17 of the human CD44 gene (FIG. 46). In some embodiments, the
extracellular domain of CD44 can be any length, which comprises
about 50 to about 100, about 100 to about 150, about 100 to about
200, about 100 to about 300, about 100 to about 400, about 100 to
about 500, about 100 to about 600, or about 100 to about 651 amino
acid residues of the extracellular domain. In some embodiments, the
extracellular domain of CD44 comprises modifications of the
polypeptide sequence. The modification can be any modification
including, but not limited to, mutations, insertions,
substitutions, deletions, and the like. In some embodiments, the
fragment comprises a mutation of Arg to Ala. In some embodiments,
the mutation of Arg to Ala occurs at a position corresponding to
position 41 in the full length CD44 protein (an example of which is
set forth in SEQ ID NO: 6). One of ordinary skill in the art can
determine which modification(s) increase the anti-cancer efficacy
of the CD44 fusion proteins when administered as either a single
agent and/or in combination with other anti-cancer therapies. One
of ordinary skill in the art can do this by, for example,
performing in vivo tumor growth and metastasis experiments to
determine the anti-cancer efficacy of the modified CD44 fusion
proteins when used as a single agent and/or in combination with
other anti-cancer therapies.
[0109] In another aspect, CD44 fusion proteins comprise different
segments of the extracellular domain of wild type CD44 or the R41A
CD44 mutant fused to another non-CD44 molecule. In some
embodiments, the non-CD44 molecule is a toxin, peptide,
polypeptide, small molecule, drug, and the like. In some
embodiments, the non-CD44 molecule is a 6-His-tag, GST polypeptide,
HA-tag, the constant region (Fc) of human IgG1, or v5-tag. In some
embodiments, the proteinase cleavage sites will be put before the
tag sequences, so that after purification these tags can be removed
by proteolytic cleavage.
[0110] Human soluble CD44-Fc (hsCD44) fusion proteins are generated
as described in the Material and Methods section of the Examples by
fusing the extracellular domain of human CD44s (SEQ ID No. 4),
CD44v3-v10 (SEQ ID No. 7+SEQ ID No. 8), CD44v8-v10 (SEQ ID No.
7+SEQ ID No. 9), CD44v6-v10 (SEQ ID No.7+SEQ ID No.20),
CD44v3-v10R41A (SEQ ID No. 6+SEQ ID No. 8), CD44v8-v10R41A (SEQ ID
No.6+No.9), CD44sR41A (SEQ ID No. 5) to the constant region (Fc) of
human IgG1. For Example, full-length CD44v3-v10 variant contains
exons 1-5, v3-v10, 16-18, and 20. CD44-v8-v10 contains exons 1-5,
v8-v10, 16-18, and 20. R41A mutation abolishes the binding capacity
of CD44 to one of its major ligands, hyaluronan (Peach et al.,
1993), and all CD44 isoforms contain this residue.
[0111] Fusion proteins between other CD44 variants and the constant
region (Fc) of human IgG1 can work effectively as potent
anti-cancer agents in similar fashion as the ones used in the
Examples. These fusion proteins include but are not limited to
CD44v4-v10-, CD44-5-v10-, CD44v7-v10-, CD44v9-v10-, CD44v10-,
CD44v3-, CD44v4-, CD44v5-, CD44v6-, CD44v7-, CD44v8-, and CD44v9-Fc
or above CD44 isoforms containing R41A mutation (Peach et al.,
1993), and different combinations and/or modifications of different
extracellular domains/exons of CD44 fused to the constant region
(Fc) of human IgG1. The modifications can be any modifications
including, but not limited to, mutations, insertions,
substitutions, deletions, and the like. The CD44 extracellular
domain can also be derived from different combinations of exons
1-17, different deletions, mutations, duplication, or
multiplication of the different segments of the extracellular
domain of CD44.
[0112] In some embodiments, the nucleic acid molecule encoding a
CD44 fusion protein is operably linked to a promoter. In some
embodiments, the promoter can facilitate the expression in a
prokaryotic cell and/or eukaryotic cell, including COS-7, CHO, 293,
human glioblastoma cells, and other human cancer cells. The
promoter can be any promoter that can drive the expression of the
nucleic acid molecule. Examples of promoters include, but are not
limited to, CMV, SV40, pEF, actin promoter, and the like. In some
embodiments, the nucleic acid molecule is DNA or RNA. In some
embodiments, the nucleic acid molecule is a virus, vector, or
plasmid. In some embodiments, the expression of the nucleic acid
molecule is regulated such that it can be turned on or off based on
the presence or absence of a regulatory substance. Examples of such
a system include, but are not limited to a tetracycline-ON/OFF
system.
[0113] Soluble recombinant CD44 HA-binding domain (CD44-HABD) was
found to block angiogenesis in vivo in chick and mouse and
inhibited growth of melanoma and pancreatic adenocarcinoma (Pall et
al., 2004). Soluble CD44 inhibits melanoma tumor growth by blocking
cell surface CD44 binding to hyaluronic acid (Ahrens et al., 2001).
CD44-receptor globulin inhibits lung metastasis of B16F10 murine
melanoma metastasis and CD44-receptor globulin contains the
extracellular part of CD44s or CD44v10 linked to the constant
region of the immunoglobulin kappa light chain (Zawadzki et al.,
1998). Soluble CD44s-immunoglobulin fusion protein inhibits in vivo
growth of human lymphoma Namalwa (Sy et al., 1992).
[0114] These CD44-Fc fusion proteins can be modified to improve
efficacy. These modifications include inserting multiple repeated
domains containing the different ligand binding sites and by fusing
the CD44 extracellular domain to the parts of the other proteins
such as the coil-coil domain of angiopoietins, which are known to
oligomerize the molecules.
CD44R41A Mutant vs. Wild Type CD44 Fusion
[0115] CD44's major ligand is hyaluronan (HA). CD44 has other
ligands such as osteopontin (Verhulst et al., 2003; Zhu et al.,
2004), fibronectin, collagen types I and IV (Ponta et al., 1998),
serglycin, laminin (Naor et al., 1997), MMP-9 (Yu and Stamenkovic,
1999, 2000), MMP-7 (Yu et al., 2002), and fibrin (Alves et al.,
2008). CD44 also cooperates with several receptor tyrosine kinases
(Orian-Rousseau and Ponta, 2008; Ponta et al., 2003), P-selectin
(Alves et al., 2008), E-selectin (Dimitroff et al., 2001; Hidalgo
et al., 2007; Katayama et al., 2005), death receptor (DR)
(Hauptschein et al., 2005), and membrane-type 1 matrix
metalloproteinase (MT1MMP) (Kajita et al., 2001).
[0116] The CD44 HA-binding site is located in the NH.sub.2-terminus
(residues 21-178). Modification of CD44 by switching R41 to A
abolishes the binding capacity of CD44 to HA (Banerji et al., 2007;
Peach et al., 1993). Therefore, modification of CD44-Fc fusion
proteins by switching R41 to A can result in CD44-Fc fusion
proteins which can effectively trap CD44 ligands other than HA.
These mutations are also likely to increase the fusion proteins'
bioavailability due to reduced sequestering of these fusion
proteins by HA in the extracellular matrix (ECM). These R41A
mutations may also result in fusion proteins with a greater
capacity for trapping other CD44 ligands and CD44 sheddase(s) due
to the increased bioavailability of CD44R41A-Fc fusion proteins,
which may be particularly important when the interaction between
CD44 and these other ligands or CD44 shedding is driving the
progression of particular types of cancer at a particular stage
and/or after a particular therapeutic treatment. We have shown that
the CD44v3-v10R41A-Fc fusion protein retained a substantial level
of anti-GBM activity (FIG. 12C-c), supporting the notion of
HA-dependent and HA-independent role of CD44 in cancer.
CD44 Fusion Protein Expression and Purification
[0117] The present invention provides an isolated nucleic acid
molecule (polynucleotide) encoding a CD44 fusion protein.
[0118] In some embodiments, the nucleic acid molecule is a
recombinant viral vector. A "recombinant viral vector" refers to a
construct, based upon the genome of a virus that can be used as a
vehicle for the delivery of nucleic acids encoding proteins,
polypeptides, or peptides of interest. Recombinant viral vectors
are well known in the art and are widely reported. Recombinant
viral vectors include, but are not limited to, retroviral vectors,
adenovirus vectors, adeno-associated virus vectors, and lenti-virus
vectors, which are prepared using routine methods and starting
materials.
[0119] Using standard techniques and readily available starting
materials, a nucleic acid molecule may be prepared. The nucleic
acid molecule may be incorporated into an expression vector which
is then incorporated into a host cell. Host cells for use in well
known recombinant expression systems for production of proteins are
well known and readily available. Examples of host cells include
bacteria cells (e.g. E. coli, yeast cells such as S. cerevisiae),
insect cells (e.g., S. frugiperda), non-human mammalian tissue
culture cells (e.g., Chinese hamster ovary (CHO) cells and Cos-7
cells), human tissue culture cells (e.g., 293 cells and HeLa
cells), glioblastoma cells, and other human cancer cells. All the
expression constructs containing nucleic acids encoding CD44 fusion
proteins, including CD44-Fc fusion proteins, contain nucleic acids
encoding the NH.sub.2-terminal signal peptide of CD44, therefore
these CD44 fusion proteins are secreted into cell culture media
(FIG. 12A, FIG. 29A, FIG. 36A, FIG. 40B, and FIG. 41B).
[0120] Some embodiments involve the insertion of DNA molecules into
a commercially available expression vector for use in well-known
expression systems. This can be accomplished using techniques known
in the art. For example, the commercially available plasmid pSE420
(Invitrogen, San Diego, Calif.) may be used for producing proteins
in E. coli. The commercially available plasmid pYES2 (Invitrogen,
San Diego, Calif.) may, for example, be used for producing proteins
in S. cerevisiae strains of yeast. The commercially available
MAXBAC.TM. complete baculovirus expression system (Invitrogen, San
Diego, Calif.) may, for example, be used for producing proteins in
insect cells. The commercially available plasmid pcDNAI, pcDNA3, or
PEF6/v5-His (Invitrogen, San Diego, Calif.) may, for example, be
used for producing proteins in mammalian cells such as Cos-7, CHO,
and 293 cells. One having ordinary skill in the art can use these
commercial expression vectors and systems or others to produce
proteins by routine techniques and readily available starting
materials. (See e.g., Sambrook et al., eds., 2001, supra) Thus, the
desired proteins or fragments can be prepared in both prokaryotic
and eukaryotic systems, resulting in a spectrum of processed forms
of the protein or fragments.
[0121] One having ordinary skill in the art may use other
commercially available expression vectors and systems or produce
vectors using well known methods and readily available starting
materials. Expression systems containing the requisite control
sequences, such as promoters and polyadenylation signals, and
preferably enhancers, are readily available and known in the art
for a variety of host cells (See e.g., Sambrook et al., eds.,
2001).
[0122] In some embodiments, the nucleic acid molecules can also be
prepared as a genetic construct. "Genetic constructs" include
regulatory elements necessary for gene expression of a nucleic acid
molecule. The elements include: a promoter, an initiation codon, a
stop codon, and a polyadenylation signal. In addition, enhancers
can be used for gene expression of the sequence that encodes the
protein or fragment. It is necessary that these elements be
operably linked to the sequence that encodes the desired
polypeptide and that the regulatory elements are operably in the
individual or cell to whom they are administered Initiation codons
and stop codon are generally considered to be part of a nucleotide
sequence that encodes the desired protein. However, it is necessary
that these elements are functional in the individual or cell to
which the gene construct is administered. The initiation and
termination codons must be in frame with the coding sequence.
Promoters and polyadenylation signals used must be functional
within the cells. Examples of promoters useful to practice the
present invention include but are not limited to promoters from
Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter,
Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal
Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV)
such as the CMV immediate early promoter, Epstein Barr Virus (EBV),
Rous Sarcoma Virus (RSV) as well as promoters from human genes such
as human Actin, human Myosin, human Hemoglobin, human muscle
creatine and human metallothionein. Examples of polyadenylation
signals useful to practice the present invention include but are
not limited to SV40 polyadenylation signals and LTR polyadenylation
signals. In some embodiments, the SV40 polyadenylation signal,
which is in the pCEP4 plasmid (Invitrogen, San Diego Calif.)
referred to as the SV40 polyadenylation signal, is used. In
addition to the regulatory elements required for DNA expression,
other elements may also be included in the DNA molecule. Such
additional elements include enhancers. The enhancer may be selected
from the group including but not limited to: human Actin, human
Myosin, human Hemoglobin, human muscle creatine and viral enhancers
such as those from CMV, RSV and EBV. Genetic constructs can be
provided with mammalian origin of replication in order to maintain
the construct extra chromosomally and produce multiple copies of
the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen
(San Diego, Calif.) contain the Epstein Barr virus origin of
replication and nuclear antigen EBNA-1 coding region which produces
high copy episomal replication without integration. In some
embodiments, the nucleic acid molecule is packaged into infectious
viral particles including but not limited to retrovirus,
adenovirus, adeno-associated virus, and lenti-virus. In some
embodiments, the nucleic acid molecule is free of infectious
particles. In some embodiments, the nucleic acid molecule is mixed
with and carried by nanoparticles.
[0123] CD44 fusion proteins are produced by the cells infected with
the expression viral constructs carrying the CD44 fusion cDNA
constructs and expressed in the presence of serum free cell culture
medium for CD44-Fc fusion proteins or 10% FBS containing medium for
all other CD44 fusion proteins. CD44 fusion proteins are purified
through affinity columns. For example, CD44-Fc fusion proteins are
purified by using protein A column as described (Sy et al., 1992)
and soluble CD44 tagged with different epitope tags are purified
using affinity column conjugated with the appropriate
antibodies.
Other CD44 Antagonists
[0124] In one aspect, the CD44 antagonist is a CD44 fusion protein.
In another aspect, the CD44 antagonist is a small molecule. In yet
another aspect, the CD44 antagonist is a shRNA or siRNA against
human CD44 (SEQ ID No. 31-38). In another aspect, shRNAs and/or
siRNAs against human CD44 are administrated in the form of a viral
vector with or without being packaged into viral particles. In one
aspect, the viral particle is a retrovirus, lentivirus, adenovirus,
or adeno-associated virus (AAV). In another aspect, the adenovirus
is a replication-impaired, non-integrating, serotype 2, 5, 6, 7, or
8 adenoviral vector. In another aspect, an shRNA against human CD44
is administrated together with other carriers including
nanoparticles. In another aspect, an shRNA against human CD44 is
administrated alone or in combination with other therapies. In
another aspect, a shRNA against human CD44 is administrated prior
to or after other anti-cancer therapies, including surgical removal
of the tumors.
DEFINITIONS
[0125] The following definitions are provided for clarity and
illustrative purposes only, and are not intended to limit the scope
of the invention.
Cancer
[0126] "Cancer" refers to abnormal, malignant proliferations of
cells originating from epithelial cell tissue (carcinomas), blood
cells (leukemias, lymphomas, and myelomas), connective tissue
(sarcomas), or glial or supportive cells (gliomas). For example,
the present invention described herein may be used for treating or
preventing malignancies of the various organ systems, such as those
affecting lung, breast, lymphoid, gastrointestinal (e.g., colon),
and genitourinary tract, prostate, ovary, pharynx, and nervous
system as well as adenocarcinomas which include but are not limited
to malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer and/or testicular tumors, non-small cell carcinoma
of the lung, cancer of the small intestine and cancer of the
esophagus. Exemplary solid tumors that can be treated include:
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, non-small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, glioblastoma multiforme (GBM), medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, and retinoblastoma, and the like. In one embodiment,
the invention relates to the treatment of renal cell carcinoma,
mesothelioma, sarcoma, or multiple myeloma. In one embodiment, the
invention relates to the treatment of colon cancer. In another
embodiment, the invention relates to the treatment of lung cancer.
In another embodiment, the invention relates to the treatment of
ovarian cancer. In an additional embodiment, the invention relates
to the treatment of breast cancer. In another embodiment, the
invention relates to the treatment of prostate cancer. In an
additional embodiment, the invention relates to the treatment of
hepatoma. In an additional embodiment, the invention relates to the
treatment of head and neck squamous carcinoma. In an additional
embodiment, the invention relates to the treatment of melanoma. In
an additional embodiment, the invention relates to the treatment of
pancreatic cancer. In yet another embodiment, the invention relates
to the treatment of astrocytomas. In a specific embodiment, the
invention relates to the treatment of gliomas, including
glioblastoma multiforme.
Cancer Stem Cells
[0127] Cancer stem cells (CSCs) or cancer initiating cells (CICs)
are a small subset of cancer cells that are capable of self-renewal
and have multi-lineage potential. These cells are responsible for
the maintenance and repopulation of tumors after therapeutic
intervention (Reya et al., 2001). CSCs are also highly resistant to
chemo- and radio-therapy, and other forms of cancer therapies. CD44
is a major cell surface marker for many types of CSCs (Stamenkovic
and Yu, 2009).
Anti-Cancer Therapies
[0128] The term "anti-cancer therapies" includes, but not limited
to, surgery, chemotherapy, radiation therapy, targeted drug
therapy, gene therapy, immunotherapy, and combination therapy that
combines at least two single therapies to treat cancers and
malignancies.
Radiation Therapy
[0129] The term "radiation therapy" or "radiotherapy" refers to use
of high-energy radiation to treat cancer. Radiation therapy
includes externally administered radiation, e.g., external beam
radiation therapy from a linear accelerator, and brachytherapy, in
which the source of irradiation is placed close to the surface of
the body or within a body cavity. Common radioisotopes used include
but are not limited to cesium (.sup.137Cs), cobalt (.sup.60Co),
iodine (.sup.131I), phosphorus-32 (.sup.32P), gold-198
(.sup.198Au), iridium-192 (.sup.192Ir), yttrium-90 (.sup.90Y), and
palladium-109 (.sup.109Pd). Radiation is generally measured in Gray
units (Gy), where 1 Gy=100 rads.
Chemotherapy and Targeted Therapy
[0130] "Chemotherapy" refers to treatment with anti-cancer drugs.
The term encompasses numerous classes of agents including
platinum-based drugs, alkylating agents, anti-metabolites,
anti-mitotic agents, anti-microtubule agents, plant alkaloids, and
anti-tumor antibiotics, kinase inhibitors, proteasome inhibitors,
EGFR inhibitors, HER dimerization inhibitors, VEGF inhibitors,
antibodies, and antisense nucleotides, siRNA, and shRNAs. Such
chemotherapeutic drugs include but are not limited to adriamycin,
melphalan, ara-C, carmustine (BCNU), temozolomide, irinotecan,
BiCNU, busulfan, CCNU, pentostatin, the platinum-based drugs
carboplatin, cisplatin and oxaliplatin, cyclophosphamide,
daunorubicin, epirubicin, dacarbazine, 5-fluorouracil (5-FU),
leucovorin, fludarabine, hydroxyurea, idarubicin, ifosfamide,
methotrexate, altretamine, mithramycin, mitomycin, bleomycin,
chlorambucil, mitoxantrone, cytarabine, nitrogen mustard,
mercaptopurine, mitozantrone, paclitaxel (Taxol.RTM.), docetaxel,
topotecan, capecetabine (Xeloda.RTM.), raltitrexed, streptozocin,
tegafur with uracil, thioguanine, thiotepa, podophyllotoxin,
filgristim, profimer sodium, letrozole, amifostine, anastrozole,
arsenic trioxide, epithalones A and B tretinioin, leustatin,
vinorelbine, vinblastine, vincristine, vindesine, etoposide,
gemcitabine, satraplatin, ixabepilone, hexamethylamine, and
thalidomide.
[0131] Targeted therapeutic agents including but not limited to
monoclonal antibodies such as Herceptin.RTM. (trastuzumab),
Rituxan.RTM. (rituximab), Campath.RTM. (alemtuzumab), Zevelin.RTM.
(Ibritumomab, tiuxetan), Alemtuzumab, Gemtuzumab, Bexxar.RTM.
(Tositumomab), ERBITUX.RTM. (Cetuximab), Bevacizumab
(Avastin.RTM.), Panitumumab (Vectibix.RTM.), Gemtuzumab
(Mylotarg.RTM.). Other targeted therapeutic agents include, but are
not limited to, tamoxifen, irinotecan, bortezomib, STI-571
(Gleevac.RTM., Imatinib Mesylate), gefitinib, erlotinib, lapatinib,
vandetanib, BIBF1120, pazopanib, neratinib, BIBW2992, CI-1033,
PF-2341066, PF-04217903, AMG 208, JNJ-38877605, MGCD-265, SGX-523,
GSK1363089, Axitinib, vatalanib, E7080, Sunitinib, Sorafenib,
Toceranib, Lestaurtinib, Semaxanib, Cediranib, Nilotinib,
Dasatinib, Bosutinib, Lestaurtinib, perifosine, MK-2206,
temsirolimus, rapamycin, BEZ235, GDC-0941, PLX-4032, imatinib,
AZD0530, bortezomib, XAV-939, advexin (Ad5CMV-p53),
Genentech--Compound 8/cIAP-XIAP inhibitor, Abbott
Laboratories--Compound 11, interleukins (e.g., 2 and 12) and
interferons, e.g., alpha and gamma, huBr-E3, Genasense, Ganite,
FIT-3 ligand, MLN491RL, MLN2704, MLN576, and MLN518. Antiangiogenic
agents include, but are not limited to, BMS-275291, Dalteparin
(Fragmin.RTM.) 2-methoxyestradiol (2-ME), thalodmide, CC-5013
(thalidomide analog), maspin, combretastatin A4 phosphate,
LY317615, soy isoflavone (genistein; soy protein isolate), AE-941
(Neovastat.TM.; GW786034), anti-VEGF antibody (Bevacizumab;
Avastin.TM.), PTK787/ZK 222584, VEGF-trap, ZD6474, EMD 121974,
anti-.alpha.v.beta.3 integrin antibody (Medi-522; Vitaxin.TM.),
carboxyamidotriazole (CAI), celecoxib (Celebrex.RTM.), halofuginone
hydrobromide (Tempostatin.TM.), and Rofecoxib (VIOXX.RTM.).
[0132] The term "gene therapy" includes administration of a vector
encoding for a CD44 fusion protein. In some embodiments, the vector
carries shRNAs against human CD44 (SEQ ID No.31-38). In some
embodiments, the vector is packaged into infectious viral particles
including, but not limited to, retrovirus, adenovirus,
adeno-associated virus, and lenti-virus. In some embodiments, the
vector is free of infectious particles. In some embodiments, the
vector is mixed with and carried by nanoparticles. Gene therapy can
include the nucleotides encoding CD44-Fc fusion, antisense
nucleotides, siRNA, and shRNAs against human CD44.
Expression Construct
[0133] The term "expression construct" means a nucleic acid
sequence comprising a target nucleic acid sequence or sequences
whose expression is desired, operatively associated with expression
control sequence elements which provide for the proper
transcription and translation of the target nucleic acid
sequence(s) within the chosen host cells. Such sequence elements
may include a promoter, an initiation codon, a stop codon, and a
polyadenylation signal. In addition, enhancers can be used for gene
expression of the sequence that encodes the protein or fragment.
The "expression construct" may further comprise "vector sequences."
By "vector sequences" is meant any of several nucleic acid
sequences established in the art which have utility in the
recombinant DNA technologies of the invention to facilitate the
cloning and propagation of the expression constructs including (but
not limited to) plasmids, cosmids, phage vectors, viral vectors,
and yeast artificial chromosomes.
[0134] Expression constructs of the present invention may comprise
vector sequences that facilitate the cloning and propagation of the
expression constructs. A large number of vectors, including
plasmid, fungal, viral vectors, have been described for replication
and/or expression in a variety of eukaryotic and prokaryotic host
cells. Standard vectors useful in the current invention are well
known in the art and include (but are not limited to) plasmids,
cosmids, phage vectors, viral vectors, and yeast artificial
chromosomes. The vector sequences may contain a replication origin
for propagation in Escherichia coli (E. coli); the SV40 origin of
replication; an ampicillin, neomycin, puromycin, hygromycin, and
blasticidin resistance gene for selection in host cells; and/or
genes (e.g., CD44-Fc fusion gene) that amplify the dominant
selectable marker plus the gene of interest. Suitable vectors,
which include plasmid vectors and viral vectors such as
bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,
vaccinia virus, semliki forest virus and adeno-associated virus
vectors, are well known and can be purchased from a commercial
source (Promega, Madison Wis.; Stratagene, La Jolla Calif.;
GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled
in the art (see, for example, Sambrook et al., eds., 2001, Meth.
Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990);
Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg.
Biomemb. 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest.
92:381-387, 1993).
Express and Expression
[0135] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription, translation, and
post-translational modification of a corresponding gene or DNA
sequence. A DNA sequence is expressed in or by a cell to form an
"expression product" such as a protein. The expression product
itself, e.g., the resulting protein, may also be said to be
"expressed" by the cell. An expression product can be characterized
as intracellular, extracellular or secreted. The term
"intracellular" means something that is inside a cell. The term
"extracellular" means something that is outside a cell. A substance
is "secreted" by a cell if it appears in significant measure
outside the cell, from somewhere on or inside the cell.
Transduction
[0136] The term "transduction" means the introduction of a
"foreign" nucleic acid (i.e. extrinsic or extracellular gene, DNA
or RNA sequence) in a viral expression vector that has been
packaged in a retro- or lenti-virus into a cell. Common techniques
in molecular biology are use to achieve virus transduction to the
appropriate cells. In one aspect, the cells are Cos-7 and 293
cells. In another aspect the cells are human GBM cells, human colon
cancer cells, human prostate cancer cells, human breast cancer
cells, human melanoma cells, human lung cancer cells, human ovarian
cancer cells, human malignant mesothelioma cells, human sarcoma
cells, human pancreatic cancer cells, human hepatoma cells, human
head and neck squamous carcinoma cells, and human multiple myeloma
cells.
Gene
[0137] The term "gene" means a DNA sequence that codes for or
corresponds to a particular sequence of amino acids which comprise
all or part of one or more proteins or enzymes, and may or may not
include regulatory DNA sequences, such as promoter and enhancer
sequences, which determine for example the conditions under which
the gene is expressed.
[0138] A coding sequence is "under the control of" or "operatively
associated with" expression control sequences in a cell when RNA
polymerase transcribes the coding sequence into RNA, particularly
mRNA, which is then trans-RNA spliced (if it contains introns) and
translated into the protein encoded by the coding sequence.
[0139] The term "expression control sequence" refers to a promoter
and any enhancer or suppression elements that combine to regulate
the transcription of a coding sequence. In a preferred embodiment,
the element is an origin of replication.
Antisense Nucleotides, siRNA, and shRNA
[0140] Antisense nucleotides are strings of RNA or DNA that are
complementary to "sense" strands of nucleotides. They bind to and
inactivate these sense strands. shRNAs are used to silence gene
expression. Antisense nucleotides can be used in gene therapy.
[0141] Small interfering RNA (siRNA) is a class of double-stranded
RNA molecules, 20-25 nucleotides in length with 2-nucleotides 3'
overhangs on either end. siRNA functions in RNA interference (RNAi)
pathway, in which it interferes with the expression of a specific
gene.
[0142] A small or short hairpin RNA (shRNA) is a sequence of RNA
that forms a tight hairpin turn that can be used to silence gene
expression via RNA interference. A shRNA usually contains two
inverted repeat sequences derived from its target gene to form
sense and antisense strand in a hairpin, which are separated by a
short spacer sequence that form a loop in shRNA and ended with a
string of T's that served as a transcription termination site. This
design produces an RNA transcript that is predicted to fold into a
short hairpin RNA. shRNA is introduced into cells using a vector
including viral vectors, which can be package into viral particles.
The vector carrying shRNAs drives their transcription by U6, H1, or
CMV (pGIPZ for shRNAmir from Open Biosystems) promoter. shRNAmir
stands for microRNA-adapted shRNA. This vector is usually passed on
to daughter cells, allowing the gene silencing to be inherited. The
shRNA hairpin is cleaved by the cellular machinery into siRNA,
which is then bound to the RNA-induced silencing complex (RISC).
This complex binds to and cleaves target mRNAs to achieve silencing
effect. siRNA and shRNA in a vector or packaged in a virus can be
used in gene therapy to knock down the expression of a gene
including that of CD44.
About or Approximately
[0143] The term "about" or "approximately" means within an
acceptable range for the particular value as determined by one of
ordinary skill in the art, which will depend in part on how the
value is measured or determined, e.g., the limitations of the
measurement system. For example, "about" can mean a range of up to
20%, preferably up to 10%, more preferably up to 5%, and more
preferably still up to 1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
5-fold, and more preferably within 2-fold, of a value. Unless
otherwise stated, the term `about` means within an acceptable error
range for the particular value.
Include or Comprise
[0144] As used herein, the terms "include" and "comprise" are used
synonymously. It should be understood that the terms "a" and "an"
as used herein refer to "one or more" of the enumerated components.
The use of the alternative (e.g., "or") should be understood to
mean either one, both, or any combination thereof of the
alternatives.
Isolated
[0145] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. Isolated nucleic acid molecules
include, for example, a PCR product, an isolated mRNA, a cDNA, or a
restriction fragment. Isolated nucleic acid molecules also include,
for example, sequences inserted into plasmids, cosmids, artificial
chromosomes, and the like. An isolated nucleic acid molecule is
preferably excised from the genome in which it may be found, and
may or may not be joined to non-regulatory sequences, non-coding
sequences, or to other genes located upstream or downstream of the
nucleic acid molecule when found within the genome. An isolated
protein may or may not be associated with other proteins or nucleic
acids, or both, with which it associates in the cell, or with
cellular membranes if it is a membrane-associated protein.
Purified
[0146] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate the
presence of unrelated materials, i.e. contaminants, including
native materials from which the material is obtained. The isolated
material is preferably substantially free of cell or culture
components, including tissue culture components, contaminants, and
the like. As used herein, the term "substantially free" is used
operationally, in the context of analytical testing of the
material. Preferably, purified material substantially free of
contaminants is at least 50% pure or 60%, 70%, 80% pure, more
preferably, 90% pure, and more preferably still at least 99% pure.
Purity can be evaluated by chromatography, gel electrophoresis,
immunoassay, composition analysis, biological assay, and other
methods known in the art.
Nucleic Acid Molecule
[0147] A "nucleic acid molecule" or "oligonucleotide" refers to the
phosphate ester polymeric form of ribonucleosides (adenosine,
guanosine, uridine or cytidine; "RNA molecules") or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine,
deoxythymidine, or deoxycytidine; "DNA molecules"), or any
phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear (e.g., restriction fragments) or circular DNA
molecules, plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0148] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, recombinant
DNA, immunology, cell biology and other related techniques within
the skill of the art. See, e.g., Sambrook et al., (2001) Molecular
Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, N.Y.; Sambrook et al., (1989) Molecular
Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, N.Y.; Ausubel et al., eds. (2005)
Current Protocols in Molecular Biology. John Wiley and Sons, Inc.:
Hoboken, N.J.; Bonifacino et al., eds. (2005) Current Protocols in
Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et
al., eds. (2005) Current Protocols in Immunology, John Wiley and
Sons, Inc.: Hoboken, N.J.; Coico et al., eds. (2005) Current
Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken,
N.J.; Coligan et al., eds. (2005) Current Protocols in Protein
Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al.,
eds. (2005) Current Protocols in Pharmacology John Wiley and Sons,
Inc.: Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression:
A Practical Approach. Oxford University Press: Oxford; Freshney
(2000) Culture of Animal Cells: A Manual of Basic Technique. 4th
ed. Wiley-Liss; among others. The Current Protocols listed above
are updated several times every year.
CD44 Fusion Compositions
[0149] In certain embodiments, the present invention relates to
pharmaceutical compositions for treating or preventing glioma or
other cancer types in a mammal. In yet another embodiment, the
present invention relates to pharmaceutical compositions for
targeting a variety of cancer stem cells in a mammal. In another
embodiment, the invention is further directed to pharmaceutical
compositions for sensitizing a variety of cancer cells and cancer
stem cells including glioma cells to radiation, cytotoxic, and
targeted therapeutic stresses for the treatment of gliomas or other
cancer types. In another embodiment, the pharmaceutical composition
comprises CD44 fusion proteins, acting as CD44 antagonists for the
treatment or prevention of a glioma or other cancer types. In
another embodiment, the pharmaceutical composition comprises a
CD44-Fc fusion protein with a constant region of human IgG1 fused
to an extracellular domain of CD44. In another embodiment, the
pharmaceutical composition comprises a CD44-Fc fusion protein with
a constant region of human IgG1 fused to the CD44 extracellular
domain of CD44s, CD44v2-v10, CD44v3-v10, CD44v8-v10, CD44v4-v10,
CD44v5-v10, CD44v6-v10, CD44v7-v10, CD44v9-v10, CD44v10, CD44v9,
CD44v8, CD44v7, CD44v6, CD44v5, CD44v4, CD44v3, CD44v2, CD44sR41A,
CD44v2-v10R41A, CD44v3-v10R41A, CD44v8-v10R41A, CD44v4-v10R41A,
CD44v5-v10R41A, CD44v6-v10R41A, CD44v7-v10R41A, CD44v9-v10R41A,
CD44v10R41A, CD44v9R41A, CD44v8R41A, CD44v7R41A, CD44v6R41A,
CD44v5R41A, CD44v4R41A, CD44v3R41A, and CD44v2R41A. In another
aspect, CD44 fusion protein comprises different segments of the
extracellular domain of wild type CD44 or R41A CD44 mutant as
described above fused to another non-CD44 molecule. In some
embodiments, the non-CD44 molecule is a toxin, peptide,
polypeptide, a small molecule, drug, and the like. In some
embodiments, the non-CD44 molecule is a 6-His-tag, GST polypeptide,
HA-tag, or v5-tag. In some embodiments, proteinase cleavage sites
will be put before the tag sequences, so that after purification
these tags can be removed by proteolytic cleavage. For example, the
HRV 3C (human rhinovirus type 14 3C) protease cleavage site
(LEVLFQ.dwnarw.GP) can be located before the COOH-terminal v5 and
His epitope tags. The HRV 3C protease specifically cleaves the
sequence LEVLFQ.dwnarw.GP at 40.degree. C. and were used to
efficiently removal the COOH-terminal tags (Novagen).
[0150] A pharmaceutical composition of a CD44 antagonist is in one
embodiment a purified CD44 fusion protein, including a purified
CD44-Fc fusion protein. In another embodiment the pharmaceutical
composition is a virus carrying a CD44 fusion protein, including a
CD44-Fc fusion protein. In yet another embodiment the
pharmaceutical composition is a siRNA/shRNA against human CD44
(e.g., SEQ ID No. 34, 35, and 36). In an additional embodiment the
pharmaceutical composition is a small molecule which antagonizes
CD44 function.
[0151] In some embodiments the glioma is an astrocytoma. In other
embodiments the glioma is a glioblastoma multiforme. In other
embodiments, the cancer types are colon cancer, breast cancer,
prostate cancer, lung cancer, ovarian cancer, pancreatic cancer,
melanoma, malignant mesothelioma, sarcoma, kidney cancer, GI track
cancer, pancreatic cancer, hepatoma, head and neck squamous
carcinoma, and multiple myeloma.
[0152] When formulated in a pharmaceutical composition, a
therapeutic compound of the present invention can be admixed with a
pharmaceutically acceptable carrier or excipient. As used herein,
the phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are generally believed to be
physiologically tolerable and do not typically produce an allergic
or similar untoward reaction, such as gastric upset, dizziness and
the like, when administered to a human.
[0153] While it is possible to use a composition provided by the
present invention for therapy as is, it may be preferable to
administer it in a pharmaceutical formulation, e.g., in admixture
with a suitable pharmaceutical excipient, diluent, or carrier
selected with regard to the intended route of administration and
standard pharmaceutical practice. Accordingly, in one aspect, the
present invention provides a pharmaceutical composition or
formulation comprising at least one active composition, or a
pharmaceutically acceptable derivative thereof, in association with
a pharmaceutically acceptable excipient, diluent, and/or carrier.
The excipient, diluent and/or carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0154] The compositions of the invention can be formulated for
administration in any convenient way for use in human or veterinary
medicine.
[0155] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as saline
solution, water, and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Alternatively, the carrier can be a solid dosage form
carrier, including but not limited to one or more of a binder (for
compressed pills), a glidant, an encapsulating agent, a flavorant,
and a colorant. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin (1990, Mack
Publishing Co., Easton, Pa. 18042).
[0156] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants, preserving, wetting, emulsifying, and dispersing agents.
The pharmaceutical compositions may be sterilized by, for example,
filtration through a bacteria retaining filter, by incorporating
sterilizing agents into the compositions, by irradiating the
compositions, or by heating the compositions. They can also be
manufactured using sterile water, or some other sterile injectable
medium, immediately before use.
[0157] In one embodiment, the pharmaceutical composition is
administered as a liquid oral formulation. Other oral dosage forms
are well known in the art and include tablets, caplets, gelcaps,
capsules, pellets, and medical foods. Tablets, for example, can be
made by well-known compression techniques using wet, dry, or
fluidized bed granulation methods.
[0158] Such oral formulations may be presented for use in a
conventional manner with the aid of one or more suitable
excipients, diluents, and carriers. Pharmaceutically acceptable
excipients assist or make possible the formation of a dosage form
for a bioactive material and include diluents, binding agents,
lubricants, glidants, disintegrants, coloring agents, and other
ingredients. Preservatives, stabilizers, dyes and even flavoring
agents may be provided in the pharmaceutical composition. Examples
of preservatives include sodium benzoate, ascorbic acid and esters
of p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used. An excipient is pharmaceutically acceptable if, in
addition to performing its desired function, it is non-toxic, well
tolerated upon ingestion, and does not interfere with absorption of
bioactive materials.
[0159] Acceptable excipients, diluents, and carriers for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington: The Science and Practice of
Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit.
2005). The choice of pharmaceutical excipient, diluent, and carrier
can be selected with regard to the intended route of administration
and standard pharmaceutical practice.
[0160] In one embodiment, the pharmaceutical compositions of CD44
antagonists are CD44 fusion proteins. In another embodiment, the
pharmaceutical compositions of CD44 antagonists are viruses
carrying an expression vector encoding CD44 fusion proteins. In
another embodiment, the pharmaceutical compositions are vectors
carrying shRNAs against human CD44 with or without being packaged
into viral particles. In one aspect, the viral particle is a
retrovirus, lentivirus, adenovirus, or adeno-associated virus
(AAV). In another aspect, the adenovirus is a replication-impaired,
non-integrating, serotype 2, 5, 6, 7, or 8 adenoviral vector.
[0161] In another embodiment, the pharmaceutical composition is
administered intravenously or intraperitoneal. In yet another
embodiment, the pharmaceutical composition is administered by
filling a cavity/space left after removal of a tumor with gel
matrix-gallocyanine formulations mixed with the pharmaceutical
composition.
[0162] In certain embodiments, the present invention relates to
methods of detecting CD44 ligands, including HA, by using CD44-Fc
fusion proteins. These methods are useful for the diagnosis and
prognosis of cancer and for the assessment of therapeutic responses
of patients.
Method of Treatment
[0163] The present invention described herein can be used to treat
cancer or malignancies. In one embodiment, the invention relates to
the treatment of prostate cancer, colon cancer, breast cancer, lung
cancer, melanoma, head-neck cancer, liver cancer, pancreatic
cancer, and ovarian cancer using CD44 fusion compositions alone or
in combinations with radiation, chemotherapy, or targeted therapy
as defined herein. In another embodiment, the invention relates to
the treatment of astrocytomas using CD44 fusion compositions alone
or in combinations with radiation, chemotherapy, or targeted
therapy. In yet another embodiment, the invention relates to the
treatment of malignant mesothelioma, sarcoma, and multiple myeloma
using CD44 fusion compositions alone or in combinations with
radiation, chemotherapy, or targeted therapy. In a specific
embodiment, the invention relates to the treatment of glioblastoma
multiforme using CD44 fusion compositions alone or in combinations
with radiation, chemotherapy, or targeted therapy. In another
specific embodiment, the invention relates to the treatment of
glioblastoma multiforme and other cancer types using CD44 fusion
compositions alone or in combinations with carmustine (BCNU),
temozolomide, docetaxel, carboplatin, cisplatin, epirubicin,
oxaliplatin, cyclophosphamide, methotrexate, fluorouracil,
vinblastine, vincristine, leucovorin, mitoxantrone, satraplatin,
ixabepilone, pacitaxel, gemcitabine, capecitabine, doxorubicin,
etoposide, melphalan, hexamethylamine, irinotecan, topotecan,
Herceptin.RTM. (trastuzumab), ERBITUX.RTM. (Cetuximab), Panitumumab
(Vectibix.RTM.), Bevacizumab (Avastin.RTM.), gefitinib, erlotinib,
lapatinib, vandetanib, neratinib, BIBW2992, CI-1033, PF-2341066,
PF-04217903, AMG 208, JNJ-38877605, MGCD-265, SGX-523, GSK1363089,
Sunitinib, Sorafenib, vandetanib, BIBF1120, pazopanib, vatalanib,
axitinib, E7080, perifosine, MK-2206, temsirolimus, rapamycin,
BEZ235, GDC-0941, PLX-4032, imatinib, AZD0530, bortezomib, XAV-939,
cIAP/XIAP inhibitors such as Compound 8 (Genentech)) (Zobel et al.,
2006) and Compound 11 (Abbott Laboratories) (Oost et al., 2004), or
advexin (Ad5CMV-p53).
[0164] "Treating" or "treatment" of a state, disorder or condition
includes: (1) preventing or delaying the appearance of clinical
symptoms of the state, disorder or condition developing in a human
or other mammal that may be afflicted with or predisposed to the
state, disorder or condition but does not yet experience or display
clinical or subclinical symptoms of the state, disorder or
condition, (2) inhibiting the state, disorder or condition, i.e.,
arresting, reducing or delaying the development of the disease or a
relapse thereof (in case of maintenance treatment) or at least one
clinical or subclinical symptom thereof, and/or (3) relieving the
disease, i.e., causing regression of the state, disorder or
condition or at least one of its clinical or subclinical
symptoms.
[0165] An "effective amount" is defined herein in relation to the
treatment of cancers is an amount that will decrease, reduce,
inhibit, or otherwise abrogate the growth of a cancer cell or tumor
by at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or at least 99%. Thus, an "effective amount" is the
quantity of compound in which a beneficial clinical outcome is
achieved when the compound is administered to a subject with a
cancer. A "beneficial clinical outcome" includes, for example, a
reduction in tumor mass, a reduction in metastasis, a reduction in
the severity of the symptoms associated with the cancer and/or an
increase in the longevity of the mammal compared with the absence
of the treatment. It will be appreciated that the amount of CD44
fusion proteins of the invention alone and/or in combinations with
chemotherapy or targeted therapy required for use in treatment will
vary with the route of administration, the nature of the condition
for which treatment is required, and the age, body weight and
condition of the patient, and will be ultimately at the discretion
of the attendant physician or veterinarian. These compositions will
typically contain an effective amount of the compositions of the
invention, alone or in combination with an effective amount of any
radiation, chemotherapy, or other targeted therapies. Preliminary
doses can be determined according to animal tests, and the scaling
of dosages for human administration can be performed according to
art-accepted practices.
[0166] The benefit to an individual to be treated is either
statistically significant or at least perceptible to the patient or
to the physician.
[0167] The present invention provides for the use of the
pharmaceutical compositions containing CD44 antagonist, such as
CD44 fusion proteins, in combination with other anti-cancer
therapies, such as but not limited to, surgery, chemotherapy,
radiation therapy, targeted therapy, and immunotherapy to treat
cancers and malignancies. In a particular embodiment of the present
invention, when combined with other anti-cancer therapies, results
in a synergistic treatment of the cancer.
[0168] The present invention is further directed to pharmaceutical
compositions and methods for sensitizing glioma and other cancer
cells to cytotoxic or targeted therapeutic stresses for the
treatment of gliomas and other cancer types. In one aspect
compositions of the present invention are administered prior to,
simultaneously with, or after other anti-cancer therapies. In
another aspect compositions of the present invention are
administered prior to or simultaneously with, or after a treatment
which causes oxidative or cytotoxic stresses. In one particular
embodiment of the invention, the stresses are caused by radiation
therapy. In another particular embodiment of the invention, the
stresses are caused by chemotherapy. In another aspect compositions
of the present invention are administered after surgical removal of
tumors.
[0169] In one aspect, the pharmaceutical compositions of CD44
antagonist, such as CD44 fusion proteins, alone or in combinations
with radiation, chemotherapy, or other targeted therapies, are
mixed with gel matrix-gallocyanine formulations and administered by
filling a cavity/space left after surgical removal of a tumor,
including a glioma. In another aspect, viruses carrying a viral
expression construct of CD44 fusion proteins or shRNAs against
human CD44, are mixed with gel matrix-gallocyanine formulations,
alone or in combination with radiation, chemotherapy or other
targeted therapies, and administered to a mammal by filling the
cavity/space left after surgical removal of a tumor, including a
glioma.
[0170] In one embodiment, the pharmaceutical composition is
administered by percutaneous injection or intralesional injection
to tumor lesions, residual tumor lesions, or adjacent normal
tissues at the surgical edge following a surgical procedure. In
another embodiment, an initial intratumoral stereotactic injection
of the pharmaceutical composition is administered 10 minutes on day
1. Patients then undergo tumor resection and receive a series of
1-minute injections of the pharmaceutical composition into the
resected tumor cavity wall on day 4. In one embodiment, the
pharmaceutical composition is administered by intralesional
injection around or near cancer tissues that cannot be surgically
removed.
[0171] For lung cancer, the pharmaceutical composition is
administrated by bronchioalveolar lavage or injected directly into
an endobronchial lesion via bronchoscopy or into locoregional
tumors via multiple percutaneous punctures under fluoroscopic,
ultrasonic, or CT scan guidance. In one embodiment, the
pharmaceutical composition is delivered to cancer lesions by CD34+
bone marrow progenitor cells, mesenchyal stem cells, or other adult
stem cells or induced pluripotent stem cells transduced to express
the pharmaceutical composition. In one embodiment, the
pharmaceutical composition is administered prior to, together with,
or after chemotherapy, radiation therapy, and other targeted
therapy.
Administration and Dosages
[0172] The CD44 fusion proteins and formulations of the present
invention can be administered topically, parenterally, orally, by
inhalation, as a suppository, or by other methods known in the art.
The term "parenteral" includes injection (for example, intravenous,
intraperitoneal, epidural, intrathecal, intramuscular,
intraluminal, intratracheal, subcutaneous, intralesional, or
intratumoral).
[0173] Administration of the compositions of the invention may be
once a day, twice a day, or more often, but frequency may be
decreased during a maintenance phase of the disease or disorder,
e.g., once every second or third day instead of every day or twice
a day. The dose and the administration frequency will depend on the
clinical signs, which confirm maintenance of the remission phase,
with the reduction or absence of at least one or more preferably
more than one clinical signs of the acute phase known to the person
skilled in the art. More generally, dose and frequency will depend
in part on recession of pathological signs and clinical and
subclinical symptoms of a disease condition or disorder
contemplated for treatment with the present compounds. For example,
the present invention can be administered intravenously or
intraperitoneally about 1-3 every week at 15 mg/kg.
[0174] Keeping the above description in mind, typical dosages of
CD44 fusion proteins may range from about 10 mg/kg to about 30
mg/kg. A preferred dose range is on the order of about 10 mg/kg to
about 15 mg/kg. In certain embodiments, a patient may receive, for
example, once per day intravenously or intraperitoneally for 8 days
each month, twice a week, or once a week.
[0175] Keeping the above description in mind, typical dosages of
viruses carrying expression vectors encoding for CD44 fusion
proteins or shRNAs against human CD44 may range from about
5.times.10e.sup.9 cfu to about 10.times.10e.sup.10 cfu. In certain
embodiments, a patient may receive a dose of viruses, for example,
by intravenous, intratumoral, or peritumoral injection once or
twice a week.
[0176] Keeping the above description in mind, typical dosages of
BCNU may range from about 50 mg/m.sup.2 to about 200 mg/m.sup.2
given iv on 3 successive days and this course being repeated at
intervals of 6 weeks (Pinkerton and Rana, 1976). A preferred dose
range is on the order of about 100 mg/m.sup.2 to about 150
mg/m.sup.2 given iv on 3 successive days and this course being
repeated at intervals of 6 weeks.
[0177] Keeping the above description in mind, typical dosages of
TMZ may range from about 50 mg/m.sup.2 to about 200 mg/m.sup.2 once
daily by intravenous infusion over 90 minutes or the oral capsule
formulation. A preferred dose range is on the order of about 75
mg/m.sup.2 to about 150 mg/m.sup.2 once daily. In certain
embodiments, a patient may receive TMZ, for example, once per day
intravenously for 5 days each month
(http://www.cancer.gov/cancertopics/druginfo/fda-temozolomide).
[0178] Keeping the above description in mind, typical dosages of
docetaxel may range from about 50 mg/m.sup.2 to about 200
mg/m.sup.2. A preferred dose range is on the order of about 60
mg/m.sup.2 to about 100 mg/m.sup.2. In certain embodiments, a
patient may receive docetaxel, for example, iv infusion once every
three weeks (http://www.drugs.com/ppa/docetaxel.html).
[0179] Keeping the above description in mind, typical dosages of
carboplatin may range from about 200 mg/m.sup.2 to about 400
mg/m.sup.2. A preferred dose range is on the order of about 300
mg/m.sup.2 to about 400 mg/m.sup.2. In certain embodiments, a
patient may receive carboplatin, for example, once intravenously
for every four weeks
(http://www.drugs.com/pro/carboplatin.html#DA).
[0180] Keeping the above description in mind, typical dosages of
cisplatin may range from about 20 mg/m.sup.2 to about 120
mg/m.sup.2. A preferred dose range is on the order of about 75 mg
to about 100 mg. In certain embodiments, a patient may receive
cisplatin, for example, once intravenously per day for 5 days every
3 wk for 3 courses.
[0181] Keeping the above description in mind, typical dosages of
cyclophosphamide may range from about 1 mg/kg/day to about 5
mg/kg/day. A preferred dose range is on the order of about 2
mg/kg/day to about 5 mg/kg/day. In certain embodiments, a patient
may receive cyclophosphamide, for example, once per day
intravenously or orally.
[0182] Keeping the above description in mind, typical dosages of
fluorouracil may range from about 12 mg/kg to about 400 mg. A
preferred dose range is on the order of about 15 mg to about 100
mg. In certain embodiments, a patient may receive fluorouracil, for
example, once of per day intravenously for 4 successive days
[0183] Keeping the above description in mind, typical dosages of
mitoxantrone may range from about 10 mg/m.sup.2 to about 20
mg/m.sup.2 given as a short iv infusion. A preferred dose range is
on the order of about 12 mg/m.sup.2 to about 14 mg/m.sup.2. In
certain embodiments, a patient may receive mitoxantrone, for
example, once intravenously every 21 days.
[0184] Keeping the above description in mind, typical dosages of
pacitaxel may range from about 3 hours at a dose of 100 mg/m.sup.2
to about 200 mg/m.sup.2. A preferred dose range is on the order of
about 3 hours at a dose of 175 mg/m.sup.2. In certain embodiments,
a patient may receive pacitaxel, for example, once intravenously
every three months.
[0185] Keeping the above description in mind, typical dosages of
topotecan may range from about 0.5 mg/m.sup.2 to about 2.5
mg/m.sup.2 daily. Topotecan can be administered by iv infused over
30 min or taking orally. A preferred dose range is on the order of
about 0.75 mg/m.sup.2-mg/m.sup.2/d.
[0186] Keeping the above description in mind, typical dosages of
trastuzumab may range from about 2 mg/kg/week to about 8
mg/kg/week. A preferred dose range is on the order of about 2 mg/kg
to about 4 mg/kg. In certain embodiments, a patient may receive
trastuzumab, for example, one intravenously every week or every
three weeks (http://www.drugs.com/ppa/trastuzumab.html).
[0187] Keeping the above description in mind, typical dosages of
cetuximab may range from about 200 mg/m.sup.2 to about 400
mg/m.sup.2. A preferred dose range is on the order of about 250
mg/m.sup.2 to about 300 mg/m.sup.2. In certain embodiments, a
patient may receive cetuximab, for example, once intravenously
every week (http://www.drugs.com/ppa/cetuximab.html).
[0188] Keeping the above description in mind, typical dosages of
panitumumab may range from about 2 mg/kg to about 10 mg/kg. A
preferred dose range is on the order of about 5 mg/kg to about 6
mg/kg. In certain embodiments, a patient may receive panitumumab,
for example, once intravenously every 14 days.
[0189] Keeping the above description in mind, typical dosages of
gefitinib may range from about 100 mg to about 400 mg. A preferred
dose range is on the order of about 250 mg. In certain embodiments,
a patient may receive gefitinib, for example, one 250 mg tablet
daily.
[0190] Keeping the above description in mind, typical dosages of
erlotinib may range from about 25 mg to about 300 mg. A preferred
dose range is on the order of about 100 mg to about 150 mg. In
certain embodiments, a patient may receive, for example erlotinib,
one tablet per day orally.
[0191] Keeping the above description in mind, typical dosages of
lapatinib may range from about 1000 mg/day to about 3000 mg/day. A
preferred dose range is on the order of about 1250 mg/day to about
1500 mg/day. In certain embodiments, a patient may receive
lapatinib, for example, one tablet per day orally.
[0192] Keeping the above description in mind, typical dosages of
BIBW2992 may range from about 20 mg/day to about 100 mg/day. A
preferred dose range is on the order of about 50 mg/day to about 70
mg/day. In certain embodiments, a patient may receive BIBW2992, for
example, once a day orally for 14 days and 14 days off for 4 weeks
(Eskens et al., 2008).
[0193] Keeping the above description in mind, typical dosages of
CI-1033 may range from about 50 mg/day to about 200 mg/day. A
preferred dose range is on the order of about 100 mg/day to about
150 mg/day. In certain embodiments, a patient may receive CI-1033,
for example, once orally over 21 consecutive days of a 28-day cycle
(Campos et al., 2005; Nemunaitis et al., 2005).
[0194] Keeping the above description in mind, typical dosages of
PF-2341066 may range from about 5 mg/kg/day to about 50 mg/kg/day.
A preferred dose range is on the order of about 20 mg/kg/day to
about 30 mg/kg/day. In certain embodiments, a patient may receive
PF-2341066, for example, once per day orally (Zou et al.,
2007).
[0195] Keeping the above description in mind, typical dosages of
sunitinib may range from about 12 mg to about 80 mg. A preferred
dose range is on the order of about 40 mg to about 50 mg. In
certain embodiments, a patient may receive sunitinib, for example,
once per day on a schedule of 4 wk on treatment followed by 2 wk
off treatment.
[0196] Keeping the above description in mind, typical dosages of
sorafenib may range from about 200 mg to about 400 mg. A preferred
dose range is on the order of about 100 mg to about 200 mg. In
certain embodiments, a patient may receive sorafenib, for example,
twice per day orally.
[0197] Keeping the above description in mind, typical dosages of
advexin (Ad5CMV-p53) may range from about 1 daily intraperitoneal
injection for ovarian cancer for 5 days every 3 weeks. Treatment
may be repeated every 21 days. For liver cancer, typical dosages of
advexin are about 1 percutaneous injection to a maximum of two
lesions on day 1. Treatment is repeated every 28 days for up to 6
courses. For breast cancer, typical dosages of advexin (Ad5CMV-p53)
are intralesional injection on days 1 and 2. Treatment repeats
every 3 weeks for up to 6 courses. For glioma, an initial
intratumoral stereotactic injection of adenovirus p53 (Ad-p53) over
10 minutes on day 1. Patients then undergo tumor resection and
receive a series of 1-minute injections of Ad-p53 into the resected
tumor cavity wall on day 4. In certain embodiments, advexin is
administrated together with or after chemotherapeutic agents or
radiation therapy.
[0198] Keeping the above description in mind, typical dosages of
Genentech--Compound 8/cIAP-XIAP inhibitor (Zobel et al., 2006) may
range from about 50 mg to about 400 mg. A preferred dose range is
on the order of about 100 mg to about 200 mg. In certain
embodiments, a patient may receive, for example 8/cIAP-XIAP
inhibitor, once per day intravenously.
[0199] Keeping the above description in mind, typical dosages of
Abbott Laboratories--Compound 11 (Oost et al., 2004) may range from
about 50 mg to about 400 mg. A preferred dose range is on the order
of about 100 mg to about 200 mg. In certain embodiments, a patient
may receive, for example Compound 11, once per day
intravenously.
[0200] Keeping the above description in mind, typical starting
dosages of epirubicin may range from about 100 to 120 mg/m.sup.2
through intravenous infusion. A preferred dose range is on the
order of about 75 mg to about 100 mg. In certain embodiments, a
patient may receive epirubicin, for example, administered
intravenously on Day 1 and repeated every 21 days for 6 cycles.
[0201] Keeping the above description in mind, typical dosages of
oxaliplatin may range from about 50 mg-200 mg/per treatment through
intravenous infusion. A preferred dose range is on the order of
about 75 mg to about 150 mg. In certain embodiments, a patient may
receive oxaliplatin, for example, administered in combination with
5-FU/LV every 2 weeks. For adjuvant use, treatment is recommended
for a total of 6 months (12 cycles. A typical treatment regiment is
the following: Day 1, oxaliplatin 85 mg/m.sup.2 IV infusion in
250-500 mL 5% Dextrose injection, USP (D5W) and leucovorin 200
mg/m.sup.2 IV infusion in D5W both given over 120 minutes at the
same time in separate bags using a Y-line, followed by 5-FU 400
mg/m.sup.2 IV bolus given over 2-4 minutes, followed by 5-FU 600
mg/m.sup.2 IV infusion in 500 mL D5W (recommended) as a 22-hour
continuous infusion. Day 2, Leucovorin 200 mg/m.sup.2 IV infusion
over 120 minutes, followed by 5-FU 400 mg/m.sup.2 IV bolus given
over 2-4 minutes, followed by 5-FU 600 mg/m.sup.2 W infusion in 500
mL D5W (recommended) as a 22-hour continuous infusion.
[0202] Keeping the above description in mind, typical dosages of
methotrexate may range from about 15 to 30 mg daily administered
orally or intramuscularly for a five-day course. Such courses are
usually repeated for 3 to 5 times as required. A preferred dose
range is on the order of about 20 mg. In certain embodiments, a
patient may receive methotrexate in combination with other
anticancer agents.
[0203] Keeping the above description in mind, typical dosages of
vinblastine is the following: initiate therapy for adults by
administering a single intravenous dose of 3.7 mg/m.sup.2 of body
surface area (bsa). A simplified and conservative incremental
approach to dosage at weekly intervals for adults may be outlined
as follows: First dose at 3.7 mg/m.sup.2 bsa, second dose at 5.5
mg/m.sup.2 bsa, third dose at 7.4 mg/m.sup.2 bsa, fourth dose at
9.25 mg/m.sup.2 bsa, and fifth dose at 11.1 mg/m.sup.2 bsa. The
above-mentioned increases may be used until a maximum dose not
exceeding 18.5 mg/m2 bsa for adults is reached. It is recommended
that the drug be given no more frequently than once every 7
days.
[0204] Keeping the above description in mind, vincristine is
administered intravenously once a week. The typical starting
dosages of vincristine for pediatric patients is 1.5-2 mg/m.sup.2
and for adults is 1.4 mg/m.sup.2.
[0205] Keeping the above description in mind, typical starting
dosages of satraplatin may range from about 100 to 120 mg/m.sup.2
once daily for 5 consecutive days every 5 weeks. A preferred dose
range is on the order of about 80 mg/m.sup.2.
[0206] Keeping the above description in mind, typical starting
dosages of ixabepilone may range about 40 mg/m.sup.2 over 3 h every
3 wk through intravenous infusion. Patients with body surface area
more than 2.2 m.sup.2 should be calculated based on 2.2 m.sup.2.
Ixabepilone may be used in combination with capecitabine.
[0207] Keeping the above description in mind, typical dosages of
gemcitabine may range about 1000 mg/m.sup.2 over 30 minutes
intravenous infusion on Days 1 and 8 of each 21-day cycle. In
certain embodiments, gemcitabine may be used in combination with
paclitaxel (breast cancer) and cisplatin (lung cancer).
[0208] Keeping the above description in mind, typical dosages of
gemcitabine may range about 1000 mg/m.sup.2 over 30 minutes
intravenous infusion on Days 1 and 8 of each 21-day cycle. In
certain embodiments, gemcitabine may be used in combination with
paclitaxel (breast cancer) and cisplatin (lung cancer). Keeping the
above description in mind, typical dosages of doxorubicin when used
as a single agent is 60 to 75 mg/m.sup.2 as a single intravenous
injection administered at 21-day intervals. The lower dosage should
be given to patients with inadequate marrow reserves due to old
age, or prior therapy, or neoplastic marrow infiltration. In
certain embodiments, doxorubicin may be used concurrently with
other approved chemotherapeutic agents. When used in combination
with other chemotherapy drugs, the most commonly used dosage of
doxorubicin is 40 to 60 mg/m2 given as a single intravenous
injection every 21 to 28 days.
[0209] Keeping the above description in mind, typical dosages of
DOXIL (doxorubicin HCl liposome injection) should be administered
intravenously at a dose of 30-50 mg/m.sup.2 at an initial rate of 1
mg/min to minimize the risk of infusion reactions. For patients
With Multiple Myeloma, Bortezomib is first administered at a dose
of 1.3 mg/m.sup.2 as intravenous bolus on days 1, 4, 8 and 11,
every three weeks. DOXIL 30 mg/m.sup.2 should be administered as a
1-hr intravenous infusion on day 4 following bortezomib.
[0210] Keeping the above description in mind, typical dosages of
etoposide (ETOPOPHOS) should be administered intravenously at a
dose of ranges from 35 mg/m.sup.2/day for 4 days to 50
mg/m.sup.2/day for 5 days. In certain embodiments, etoposide may be
used in combination with other anticancer agents.
[0211] Keeping the above description in mind, typical dosages of
melphalan (ALKERAN Tablets) should be administered orally at a dose
about 6 mg (3 tablets) daily. After 2 to 3 weeks of treatment, the
drug should be discontinued for up to 4 weeks. In certain
embodiments, melphalan may be used in combination with other
anticancer agents including bortezomib.
[0212] Keeping the above description in mind, hexamethylamine
(Hexylen, Altretamine, Hexastat) as HEXALEN.RTM. capsules is
administered orally. Doses are calculated on the basis of body
surface area. HEXALEN.RTM. capsules may be administered either for
14 or 21 consecutive days in a 28 day cycle at a dose of 260
mg/m.sup.2/day. The total daily dose should be given as 4 divided
oral doses after meals and at bedtime. HEXALEN.RTM. capsules should
be temporarily discontinued (for 14 days or longer) and
subsequently restarted at 200 mg/m.sup.2/day.
[0213] Keeping the above description in mind, irinotecan
(CAMPTOSAR) may be used either as a single agent or in combination
with fluorouracil and leucovorin at a dosage of 125 mg/m.sup.2
intravenously over 90 minutes once a week for four doses or as a
single agent at a dosage of 350 mg/m.sup.2 intravenously over 90
minutes every three weeks, or in combination with fluorouracil and
leucovorin at a dosage of 180 mg/m.sup.2 intravenously over 90
minutes every other week for three doses.
[0214] Keeping the above description in mind, typical dosages of
PF-04217903 may range from about 50 mg to about 1000 mg
administrating orally twice a day. A treatment cycle is considered
to be 21 days. A preferred dose range is on the order of about 100
mg to about 500 mg.
[0215] Keeping the above description in mind, typical dosages of
AMG 208 may range from about 10 mg to about 1000 mg administrating
orally twice a day. A preferred dose range is on the order of about
100 mg to about 500 mg.
[0216] Keeping the above description in mind, typical dosages of
JNJ-38877605 may range from about 10 mg to about 1000 mg
administrating orally once or twice a day. A treatment cycle is
considered to be 21 days. A preferred dose range is on the order of
about 100 mg to about 500 mg.
[0217] Keeping the above description in mind, typical dosages of
MGCD-265 may range from about 24 mg/m2 to about 340 mg/m2
administrating orally and daily with 7 days on/7 days off schedule
for a 28-day cycle. A preferred dose range is on the order of about
200 mg to about 500 mg.
[0218] Keeping the above description in mind, typical dosages of
SGX-523 may range from about 10 mg to about 500 mg administrating
orally twice a day on a 14 days on/7 days off therapy schedule,
cycling every 21 days. A preferred dose range is on the order of
about 100 mg to about 200 mg.
[0219] Keeping the above description in mind, typical dosages of
GSK1363089 may range at about 240 mg/d on day 1-5 repeated every 14
days with 5 day on/9 day off schedule or at about 80 mg/d daily.
The drug will be administrated orally. A preferred dose range is on
the order of about 80 mg to about 200 mg.
[0220] Keeping the above description in mind, typical dosages of
vandetanib may range from about 100 mg to about 500 mg
administrating orally once a day. A preferred dose range is on the
order of about 100 mg to about 300 mg.
[0221] Keeping the above description in mind, typical dosages of
BIBF1120 may range from about 100 mg to about 250 mg administrating
orally twice a day in a 20-day continuous dosing regimen. A
preferred dose range is on the order of about 100 mg to about 200
mg.
[0222] Keeping the above description in mind, the recommended dose
of VOTRIENT (pazopanib) may range from about 200 mg to about 800 mg
orally once daily without food (at least 1 hour before or 2 hours
after a meal).
[0223] Keeping the above description in mind, typical dosages of
bevacizumab may range from about 5 mg-10 mg/kg every 2 weeks; 5
mg/kg or 10 mg/kg every 2 weeks when used in combination with
intravenous 5-FU-based chemotherapy; about 15 mg/kg every 3 weeks
in combination with carboplatin and paclitaxel; about 10 mg/kg
every 2 weeks in combination with interferon alfa; and about 10
mg/kg every 2 weeks in combination with paclitaxel. Bevacizumab
should be administrated through intravenous (IV) infusion over 90
minutes in a 20-day continuous dosing regimen. A preferred dose
range is on the order of about 100 mg to about 200 mg.
[0224] Keeping the above description in mind, typical dosages of
vatalanib may range from about 250 mg to about 2000 mg
administrating orally daily in a 28-day continuous dosing regimen.
A preferred dose range is on the order of about 1000 mg to about
1500 mg.
[0225] Keeping the above description in mind, typical dosages of
axitinib may range from about 5 mg to about 30 mg twice daily
administrating orally daily. A preferred dose range is on the order
of about 5 mg to about 10 mg.
[0226] Keeping the above description in mind, typical dosages of
E7080 may range from about 0.1 mg-12 mg administrating orally
continually twice daily for 2-6 cycles of a 28-day cycle. A
preferred dose range is on the order of about 5 mg to about 10
mg.
[0227] Keeping the above description in mind, typical dosages of
perifosine may range from about 100-600 mg/week administrating
orally. A preferred dose range is on the order of about 200 mg to
about 400 mg.
[0228] Keeping the above description in mind, typical dosages of
MK-2206 may range from about 30 mg-60 mg administrating orally
every other day in a 28-day cycle. A preferred dose range is on the
order of about 30 mg to about 50 mg.
[0229] Keeping the above description in mind, typical dosages of
temsirolimus may range from about 25 mg-about 50 mg administrating
through infused over a 30-60 minute period once a week. A preferred
dose range is on the order of about 30 mg.
[0230] Keeping the above description in mind, typical dosages of
rapamycin may range from about 10 mg-40 mg administrating orally
daily. A preferred dose range is on the order of about 20 mg to
about 30 mg.
[0231] Keeping the above description in mind, typical dosages of
BEZ235 may range from about 10 mg-45 mg administrating orally once
daily on days 1-28 of the first course. Courses will repeat every
28 days. A preferred dose range is on the order of about 20 mg to
about 30 mg.
[0232] Keeping the above description in mind, typical dosages of
GDC-0941 may range from about 60 mg-80 mg administrating orally
once daily or twice a day. A preferred dose range is on the order
of about 40 mg to about 50 mg.
[0233] Keeping the above description in mind, typical dosages of
PLX-4032 may range from about 200 mg-960 mg administrating orally
twice daily. A preferred dose range is on the order of about 300 mg
to about 500 mg.
[0234] Keeping the above description in mind, typical dosages of
imatinib may range from about 400 mg-800 mg administrating orally
daily or twice daily. A preferred dose range is on the order of
about 400 mg to about 500 mg.
[0235] Keeping the above description in mind, typical dosages of
AZD0530 may range from about 100 mg-500 mg/week administrating
orally. A preferred dose range is on the order of about 100 mg to
about 250 mg.
[0236] Keeping the above description in mind, typical dosages of
VELCADE (bortezomib) is 1.3 mg/m2 administered as a 3 to 5 second
bolus IV injection in combination with oral melphalan and oral
prednisone for nine 6-week treatment cycles. In cycles 1 through 4,
bortezomib is administered twice weekly (days 1, 4, 8, 11, 22, 25,
29 and 32). In cycles 5 through 9, bortezomib is administered once
weekly (days 1, 8, 22 and 29). At least 72 hours should elapse
between consecutive doses of bortezomib.
[0237] Keeping the above description in mind, typical dosages of
XAV-939 may range from about 100 mg-500 mg/week administrating
orally. A preferred dose range is on the order of about 100 mg to
about 250 mg.
[0238] Keeping the above description in mind, the dosage of the
chemotherapeutic agent or cytotoxic drug may be less than that
normally used when administered in combination with the CD44-Fc
fusion protein, as described herein these fusion proteins
sensitizes cancer cells to cytotoxic drugs.
EXAMPLES
[0239] The present invention is described further below in working
examples which are intended to further describe the invention
without limiting the scope therein.
Materials and Methods
[0240] In the examples below, the following materials and methods
were used.
Patient Glioma Samples
[0241] The glioma tissues were obtained from Cooperative Human
Tissue Network (CHTN) at University of Pennsylvania and The Ohio
State University. Human tissues were used in accordance with the
approved Human tissue study protocol.
Expression Data Mining
[0242] The Oncomine database (www.oncomine.org, Compendia
Bioscience, Ann Arbor, Mich.) was searched for CD44 mRNA expression
levels in human glioma tissues and other human cancer types
compared to their normal counterparts.
Expression Profiling and Real-Time Quantitative PCR (qPCR)
[0243] To compare gene expression profiles, human U133v2 gene chips
(Affymetrix) were used and the probes derived from three
independently transduced and pooled puromycin-resistant U87MG or
WM793 cells that re-express merlin (U87MG/Wm793merlin) or were
transduced with empty retroviruses (U87MG/WM793 wt) following
standard protocols.
Cell Lines and Reagents
[0244] Human glioma cells, U138MG, LN118, LN229, and A172 cells
(ATCC); SNB19, SNB75, SNB78, U118MG, U87MG, U251, U373MG, SF763,
SF767, SF268, SF539, SF188, SF295, and SF242 (UCSF and NCI), and
normal human astrocytes (NHAs, ALLCELLS, Inc) were maintained
according to the providers' and manufacturers' instructions.
Anti-MST1/2, -Lats1/2 (Bethyl Lab), -CD44, -Erk1/2, -AKT, -JNK,
-p21, -p38, -p53, -cIAP1/2, and -merlin (Santa Cruz), -actin
(Sigma), -nestin (Millipore), -sox-2 (R&D systems), -v5
epitope, -phospho-merlin, -puma (Invitrogen), -cleaved caspase 3,
-phospho-Erk1/2, -phospho-AKT, -phospho-JNK, -phospho-p38,
-phospho-MST1/2, -phospho-Lats1, -phospho-YAP (Cell signaling),
-YAP, -phospho-Lats2 (Abnova) and -heparan sulfate (HS, CalBiochem)
antibodies were used in the experiments. Apoptag kit was from
Chemicon and anti-Brdu from Roche.
Establishment of Primary Human Glioma, Lung, Breast, and Ovarian
Cancer Spheres
[0245] Fresh human glioblastoma, lung cancer, prostate cancer,
breast cancer, ovarian cancer, and melanoma tissues were obtained
from Cooperative Human Tissue Network (CHTN) at University of
Pennsylvania and The Ohio State University. The tissues were
dissociated into single cells by 0.4% collagenase type I (Sigma
C0130) and plated in ultra-low attachment plates in serum-free
cancer stem cell culture medium, which is DMEM/F12 supplemented
with B27 (Invitrogen), EGF (10 ng/mL, BD Biosciences), and FGF-2
(20 ng/mL, BD Biosciences). After formation of the initial spheres,
cancer spheres, including glioma spheres, were passaged
approximately every-two week by dissociating the spheres with 0.05%
trypsin-ethylenediamine tetraacetic acid (EDTA).
Engineering CD44-Fc Fusion Expression Vectors and Knockdown
Constructions
[0246] Human total spleen RNAs were obtained from Clontech. Total
RNAs from human skin tissues (CHTN-University of Pennsylvania) and
T47D human breast cancer cells (ATCC) were isolated using RNeasy
Mini Kit (Qiagen) according to the manufacturer's instructions.
cDNA was synthesized from 5 .mu.g of total RNA using Superscript II
RNase H.sup.- reverse transcriptase (Invitrogen). Human Fc fragment
was obtained by PCR using human spleen cDNAs as templates, Pfu DNA
polymerase (Stratagene), and a pair of primers as the following:
forward primer, 5'-gacaaaactcacacatgcccaccg-3' (SEQ ID NO. 71) and
reverse primer, 5' tcatttacccggagacagggagag-3' (SEQ ID NO. 72).
Human skin and T47D human breast cancer cells expressing many CD44
isoforms including human CD44v3-v10, CD44v8-v10, and CD44s were
obtained. Human soluble CD44 isoforms were obtained by PCR using
mixture of human skin and T47D cDNAs as templates, Pfu DNA
polymerase (Stratagene), a pair of primers as the following:
forward primer, 5'-acc atg gac aag ttt tgg tgg cac-3' (SEQ ID NO.
73) and reverse primer, 5'-ttctggaatttggggtgtccttat-3' (SEQ ID NO.
74). All the resulting PCR products were cloned into
pEF6/v5-HisTOPO expression vectors (Invitrogen). The clones with
correct human Fc fragment and soluble CD44 were identified. These
fragments were then subcloned into the retroviral expression vector
pQCXIP (BD Bioscience) to generate human soluble CD44-Fc (hsCD44)
fusion expression constructs. The soluble human CD44v3-v10, v8-v10,
or soluble CD44s were fused in frame to the human Fc fragment using
a MfeI restriction site (CAATTG). Retroviruses were generated using
these expression constructs and pVSVG/GP2 in 293 cells following
the manufacturer's instructions (BD). All expression constructs
were verified by DNA sequencing.
Deletion and Point Mutagenesis
[0247] The following soluble human CD44-Fc fusion protein
constructs have been generated CD44s-, CD44v3-v10-, CD44v8-v10-,
CD44v4-v10-, CD44v6-v10-, CD44v7-v10-, CD44v9-v10-, and CD44v10-Fc.
The following soluble human CD44-Fc fusion protein constructs are
being generated by deletional mutagenesis: CD44v5-v10-, CD44v9-,
CD44v8-, CD44v7-, CD44v6-, CD44v5-, CD44v4-, and CD44v3-Fc.
Deletional mutagenesis is performed by using soluble human
CD44v3-v10-Fc in the retroviral expression vector pQCXIP (BD
Bioscience) as the template together with the ExSite mutagenesis
kit (Stratagene), and different pairs of appropriate primers
corresponding to the sequences of 24 nucleotides before and after
the segments intended to be deleted as described (Bai et al.,
2007).
[0248] The following soluble human CD44R41A-Fc mutated fusion
protein constructs have been generated: CD44sR41A-,
CD44v8-v10R41A-, and CD44v3-v10R41A-Fc. The following soluble human
CD44R41A-Fc mutated fusion protein constructs will be generated by
point mutation: CD44v4-v10R41A-, CD44v5-v10R41A-, CD44v6-v10R41A-,
CD44v7-v10R41A-, CD44v9-v10R41A-, CD44v10R41A-, CD44v9R41A-,
CD44v8R41A-, CD44v7R41A-, CD44v6R41A-, CD44v5R41A-, CD44v4R41A-,
CD44v3R41A-Fc. The point mutation in CD44sR41A-, CD44v8-v10R41A-,
and CD44v3-v10R41A-Fc were generated by using soluble human CD44s-,
CD44v8-v10-, CD44v3-v10-Fc in retroviral expression vector pQCXIP
(BD Bioscience) as the templates together with the QuikChange.RTM.
II Site-Directed Mutagenesis Kit (Stratagene), and a pairs of
appropriate primers: forward, 5'-gtg gag aaa aat ggt gcc tac agc
atc tct cgg-3' (SEQ ID NO. 75) and reverse, 5'-ccg aga gat get gta
ggc acc att ttt etc cac-3' (SEQ ID NO. 76). The retroviruses were
generated by using these expression constructs and pVSVG/GP2-293
cells following the manufacturer's instructions (BD Bioscience).
Similar procedures will be used to generate additional CD44R41A-Fc
constructs.
Produce and Purify Soluble CD44-Fc and Soluble CD44R41A-Fc Fusion
Proteins
[0249] Cos-7 cells infected with the retroviruses carrying
hsCD44v3-v10-Fc, hsCD44v6-v10-FC, hsCD44v8-v10-Fc, hsCD44s-Fc,
hsCD44v3-v10R41A-Fc, hsCD44v8-v10R41A-Fc, and hsCD44sR41A-Fc
constructs were cultured in RPMI medium containing 10% fetal bovine
serum (FBS) to reach confluence then switched to serum free RPMI
medium (SFM) to culture for additional three days. The collected
SFM was purified through protein A columns (GE Healthcare
Biosciences). Before elution from protein A column, some of
preparations of the bound CD44-Fc fusion proteins were treated
heparinase I (10 units/ml) and heparinase III (2 unit/ml) or at
37.degree. C. for 4 h.
Luciferase Reporter Assay
[0250] To measure canonical Wnt signaling in U87MGwt and
U87MGmerlinS518D, U87MGmerlin, and U87MGmerlinS518A cells, the
beta-catenin-responsive luciferase reporter construct (TopFlash,
Addgene), which contains TCF/LEF binding sites and a negative
control construct, FopFlash, which contains mutated TCF/LEF binding
sites, was used. These reporters were transfected transiently into
these transduced glioma cells in triplicate. The luciferase
activity in these transfected cells were measured 24 hours
post-transfection following the manufacturer's instructions
(Promega) using a Modulus Microplate Luminometer/Fluorometer
(Turner Biosystems).
[0251] To knock down human CD44 expression, several shRNAmir
(expression Arrest.TM. microRNA-adapted shRNA) and TRC (the RNAi
consortium) constructs against human CD44 and a non-targeting
shRNAmir and non-targeting TRC control constructs were obtained
from Open Biosystems and Addgene (a non-profit plasmid repository,
www.addgene.org). Lentiviruses carrying these shRNAs were generated
following the manufacturer's instructions. Expression Arrest.TM.
microRNA-adapted shRNA (shRNAmir) are designed to mimic a natural
microRNA primary transcript, enabling specific processing by the
endogenous RNAi pathway and producing more effective knockdown.
microRNA-30 adapted design contains mir-30 loop and context
sequences (Silva et al., 2005)
TABLE-US-00002 TABLE 2 Sequence Listings for the CD44 Antisense
Constructs nucleotides Sequence SEQ ID No. shRNA-TRC- sense loop
antisense: 31 CD44#1 GCCCTATTAGTGATTTCCAAA CTCGAG
TTTGGAAATCACTAATAGGGC shRNA-TRC- sense loop antisense: 32 CD44#2
CGGAAGTGCTACTTCAGACAA CTCGAG TTGTCTGAAGTAGCACTTCCG shRNA-TRC- sense
loop antisense: 33 CD44#3 CCTCCCAGTATGACACATATT CTCGAG
AATATGTGTCATACTGGGAGG shRNA-TRC- sense loop antisense: CD44#4
CCAACTCTAATGTCAATCGTT CTCGAG 34 AACGATTGACATTAGAGTTGG shRNA-TRC-
sense loop antisense: CD44#5 CGCTATGTCCAGAAAGGAGAA CTCGAG 35
TTCTCCTTTCTGGACATAGCG shRNAmir- mir-30 context sequence sense loop
antisense mir-30 36 CD44#1 context sequence: TGCTGTTGACAGTGAGCG
AGGTGTAACACCTACACCATTA TAGTGAAGCCACAGATGTA TAATGGTGTAGGTGTTACACCC
TGCCTACTGCCTCGGA shRNAmir- mir-30 context sense loop antisense
mir-30 context: 37 CD44#2 TGCTGTTGACAGTGAGCG ACGCAGATCGATTTGAATATAA
TAGTGAAGCCACAGATGTA TTATATTCAAATCGATCTGCGC TGCCTACTGCCTCGGA
shRNAmir- mir-30 context sense loop antisense mir-30 context: 38
CD44#3 TGCTGTTGACAGTGAGCG CCCTCCCAGTATGACACATATT
TAGTGAAGCCACAGATGTA AATATGTGTCATACTGGGAGGT TGCCTACTGCCTCGGA
shRNA-TRC-NT CCGCAGGTATGCACGCGT (Addgene) 39 shRNAmir-NT mir-30
context sense loop antisense mir-30 context: 40 TGCTGTTGACAGTGAGCG
ACCTCCACCCTCACTCTGCCAT TAGTGAAGCCACAGATGTA ATGGCAGAGTGAGGGTGGAGGG
TGCCTACTGCCTCGGA
[0252] Lenti- and Retroviral Transduction
[0253] U87MG and U251 human glioma cells were seeded in 6-well
plates and allowed to grow for overnight. The subconfluence U87MG,
and U251 cells were first transduced with the retroviruses carrying
luciferase with a hygromycin-resistant gene, and then transduced
with the retroviruses carrying the empty retroviral expression
vector or human soluble (hs) CD44-Fc fusion constructs with a
puromycin-resistant gene. The pooled populations of drug resistant
cells were expanded, and portions of the cells were used to assess
their expression of the transduced gene products. Anti-CD44 and
anti-human IgG antibodies were used to detect the expression level
of hsCD44-Fc fusion proteins.
[0254] CD44 knockdown was accomplished using lentiviruses carrying
shRNAs against human CD44 or non-targeting control shRNAs following
the manufacturer's instructions. Infected cells were selected for
their resistance to hygromycin and puromycin. The pooled
populations of the drug resistant cells were expanded and portions
of the cells were used to assess the expression level of endogenous
CD44. Anti-CD44 antibodies (Santa Cruz) were used for assessing
endogenous level of CD44.
Glioma Sphere Transduction
[0255] Human glioma spheres (HGSs) were disaggregated with 0.05%
trypsin-ethylenediamine tetraacetic acid (EDTA, Cellgro.RTM.) and
seeded on the BD BioCoat.TM. Matrigel.TM. Matrix 6-well plates,
which are designed to maintain and propagate embryonic stem cells
in the absence of feeder layers. These cells were transduced with
lentiviruses carrying shRNAs against human CD44. After selection
with puromycin, the pooled populations of drug-resistant cells were
suspended into single cells and cultured in serum-free cancer stem
cell culture medium (DMEM/F12 supplemented with B27 (Invitrogen),
EGF (10 ng/mL, BD Biosciences), and FGF-2 (20 ng/mL, BD
Biosciences)) in ultra-low attachment plates to re-form
spheres.
Western Blot Analysis of CD44 Expression
[0256] Cells were extracted with either RIPA buffer (50 mM Tris-HCl
(pH7.4) containing 150 mM NaCl, 5 mM EDTA, 1% Triton, 0.1% SDS, 2
mM PMSF, 2 .mu.g/ml leupeptin, and 0.05 U/ml aprotinin) or with
4.times.SDS Laemmli sample buffer without the dye and protein
concentrations were determined using Bio-Rad Dc Protein Assay
Reagents. 50-100 .mu.g of extracted proteins were separated by 10%
SDS-PAGE. Following electrophoresis, the gels were blotted onto
Hybond-ECL membranes (Amersham, Arlington Heights, Ill.). Anti-CD44
antibody (Santa Cruz) was employed to detect CD44.
Immunocytochemistry of CD44 Expression
[0257] Glioma cells with or without CD44 knockdown were cultured in
35 mm dishes in the presence of 10% FBS RPMI for 24 hours. The
cells were fixed in 3.7% paraformaldehyde, washed with PBS, and
blocked with 2% non-fat milk. Anti-CD44 antibody (Santa Cruz) and
FITC-conjugated anti-mouse secondary antibody (Sigma) were employed
to detect cell surface CD44.
Fluorescein-Labeled HA (FL-HA) Binding Assay
[0258] FL-HA binding assay was performed as described previously
(Xu and Yu, 2003; Yu and Stamenkovic, 1999). Briefly, a total of
5.times.10.sup.5 of the transduced glioma cells were seeded onto
35-mm dishes in the presence of PRMI/10% FBS and puromycin. On the
following day, the culture medium was replaced by fresh RPMI/10%
FBS containing 20 .mu.g/ml Fl-HA. Twenty-four later, the cells were
washed extensively with PBS, fixed in 4% paraformaldehyde, washed,
mounted, and observed under a fluorescence microscope.
Subcutaneous Tumor Growth Experiments
[0259] Mice were used in accordance with the approved IACUC
Protocol. Pooled populations of transduced U87MG and U251 glioma
cells were used for subcutaneous tumor growth experiments. 2 or
5.times.10.sup.6 glioma cells or 5.times.10.sup.6 of glioma cells
were injected subcutaneously into each immuno-compromised
B6.129S7-Rag1.sup.tmMom (Rag1, Jackson Lab) mouse. Six mice were
used for each type of the infected glioma. After solid tumors
became visible (10-15 days after the injection), the longest and
shortest diameters of the solid tumors were measured using a
digital caliper every third day for five to seven weeks for
gliomas. Tumor volumes were calculated using the following formula:
tumor volume=1/2.times.(shortest diameter).sup.2.times.longest
diameter (mm.sup.3). At end of the experiments, tumors were fixed
and sectioned for histological and immunohistochemical
analyses.
Intracranial Tumor Growth Experiment
[0260] Mice were used in accordance with the approved IACUC
Protocol. Pooled populations of the transduced U87MG and U251 cells
were used for the intracranial tumor growth experiments. U87MG
(4.times.10.sup.5 cells in 10 .mu.l HBSS/Rag1 mouse)/U251 cells
(2.times.10.sup.5 cells in HBSS/Rag1 mouse) were injected at the
bregma 2 mm to the right of the sagittal suture and 3 mm below the
surface of the skull. Following injection, mice were closely
monitored and the duration of their survival was recorded. Mice
that showed signs of distress and morbidity were euthanized and
considered as if they had died on that day. Number of surviving
mice was recorded. The survival rates were calculated as follows:
survival rate (%)=(number of mice still alive/total number of
experimental mice).times.100%. Mice that were free of symptoms 40
or 60 days after intracranial injection were euthanized and the
tissues examined.
Bioluminescence Imaging Analysis of the Intracranial Gliomas
[0261] To monitor the growth of intracranial gliomas in live
animal, bioluminescence-imaging approach was used. U87MG and U251
cells were infected with a retroviral-based luciferase expression
vector that contains an internal ribosome entry site (IRES) and
hygromycin resistance gene. Hygromycin-resistant U87MG-Luc and
U251-Luc cells express high levels of luciferase. These cells were
then infected with lentiviruses carrying non-targeting shRNAs or
shRNAs against human CD44. These double drug resistant cells were
injected intracranially into Rag-1 mice at the bregma 2 mm to the
right of the sagittal suture and 3 mm below the surface of the
skull. 3, 6, 9, 13, 17 days after the injections, bioluminescence
images of the intracranial tumors were acquired 12 min after
injection of D-luciferin using the same intensity scaling by using
IVIS-200 imaging system (Xenogen) at the In Vivo Molecular Imaging
Shared Facility at Mount Sinai School of Medicine.
Histology and Immunohistochemistry
[0262] To determine the glioma cell proliferation rate in vivo,
5-Bromo-2'-deoxy-uridine (BrdU) was injected intraperitoneally
(i.p.) into mice four hours prior to euthanasia. Tumors including
gliomas from the experimental animals were dissected and fixed in
formalin (Fisher), washed with PBS, dehydrated through 30%, 70%,
95%, and 100% ethanol and xylene, and embedded in paraffin wax
(Fisher). 5-10 .mu.m sections were cut, mounted onto slides and
stained with hematoxylin and eosin (Fisher) for histologic
analysis. The sections were incubated with anti-BrdU or anti-Ki67
antibodies to detect proliferating cells or with the Apoptag kit to
detect apoptotic cells in situ (Lau et al. 2008).
Western Blot Analysis of Signaling Pathway Proteins
[0263] U87MG-NT cells (U87MG cells infected with a mixture
lentiviruses carrying non-targeting TRC-NT and shRNAmir-NT
constructs) and U87MGshRNA-CD44 cells (U87MG cells infected with a
mixture lentiviruses carrying shRNAs against human CD44, TRC-CD44#3
and shRNAmir-CD44#1) were treated with vehicle, 60 .mu.m
H.sub.2O.sub.2 or 40 .mu.g/ml TMZ for 30 min, 2 h, 24 h, 48 h, and
72 h. The cells were lysed using 4.times.SDS Laemmli sample buffer
without the dye. Protein concentrations were determined using
Bio-Rad Dc Protein Assay Reagents. 100 .mu.g of total protein was
loaded in each lane. Actin was included as an internal control for
protein loading. The antibodies used against the different
signaling mediators are indicated in the figures.
[0264] Western blots were also performed using cell lysates derived
from U87MG-TN and U87MGshRNA-CD44 cells treated with different
growth factors. 2.times.10.sup.5 of the glioma cells were seeded
into 6-well plates for 24 hours and switched to serum free medium
and cultured for additional 72 hours. The serum starved U87MG cells
were treated with or without FBS, NGF (10 ng/ml), EGF (2 ng/ml),
HB-EGF (5 ng/ml), betacellulin (BTC, 5 ng/ml), epiregulin (Epr, 5
ng/ml), amphiregulin (AR, 5 ng/ml), or HGF (20 ng/ml) for 12 h. The
cells were lysed using 4.times.SDS Laemmli sample buffer without
the dye and protein concentrations were determined using Bio-Rad Dc
Protein Assay Reagents. 100 .mu.g of total protein were loaded in
each lane. Actin was included as an internal control for protein
loading. The antibodies used against the different signaling
mediators are indicated in the figures.
Administration of Oxidative Stress
[0265] H.sub.2O.sub.2 was added into serum-free glioma culture
medium (RPMI) to reach a final concentration of 60 .mu.M. The
glioma cells were cultured in the presence of 60 .mu.m
H.sub.2O.sub.2 for 30 min, 2 h, 24 h, 48 h, and 72 h.
Administration of Chemotherapeutic Agents
[0266] TMZ was added into the serum-free glioma culture medium
(RPMI) to reach a final concentration of 40 .mu.g/ml. The glioma
cells were cultured in the presence of 40 .mu.g/ml TMZ for 30 min,
2 h, 24 h, 48 h, and 72 h.
Methods of Detecting HA Using Biotin-Labeled CD44-Fc Fusion
Proteins and Methods of Diagnosing Cancers by Detecting HA
[0267] Purified CD44-Fc fusion proteins (hsCD44s-Fc,
hsCD44v8-v10-Fc, and hsCD44v3-v10-Fc) were labeled with biotin
using EZ-Link Biotinylation Kits (Thermo Scientific) following the
manufacturer's instruction. Human tumor paraffin sections were
deparaffinized and rehydrated. After blocking with 2% BSA, the
sections were incubated with biotinylated CD44-Fc fusion proteins
(1 .mu.g/ml) for overnight at 4 degree. biotinylated CD44-Fc fusion
proteins were detected by VECTASTAIN ABC kit.
[0268] To detect plasma HA level, at least 200 .mu.l blood from
each transgenic mice (MMTV-PyVT and MMTV-ActErbb2, Jackson Lab)
bearing breast cancer, Rag-1 mice bearing gliomas derived from
MSSM-GBMCSC-1 or Glioma 261 cells, or control health mice were
collected. Blood samples from six mouse of each type of mice were
collected and plasma samples were generated immediately. 50 .mu.l
plasma from each sample was loaded in triplicate into each well of
an Elisa plate that has been pre-coated with CD44-Fc fusion
proteins. The CD44-Fc bound HA was detected by biotinylated CD44-Fc
fusion proteins and AP-conjugated avidin. The developed color was
measure by an Elisa machine at 405 nm.
Prostate, Colon, Breast, Lung, Ovarian, Liver, Pancreatic, and
Head-Neck Cancer Models and Melanoma Model
[0269] PC3/M human prostate cancer cells, HCT116 and KM20L2 human
colon cancer cells, MX-2 and SW613 human breast carcinoma cells,
NCI-H125 human non-small cell lung cancer cells, NCIH460, human
large cell lung cancer cells, and OVCAR-3 human ovarian cancer
cells were transduced with luciferases and shRNAs against human
CD44 or control non-targeting shRNAs and selected for their
resistance to hygromycin and puromycin. M14 human melanoma cells,
SCC-4 human head-neck carcinoma cells, BXPC-3 human pancreatic
cancer cells, and SK-Hep-1 human liver cancer cells were transduced
with retroviruses carrying CD44s-Fc, CD44v3-v10-Fc, and
CD44v8-v10-Fc constructs or empty expression vector. Pooled
populations of the drug-resistant cancer cells were used for
subcutaneous tumor growth experiments. 5.times.10.sup.6 of these
cancer cells were injected subcutaneously into each
immuno-compromised B6.129S7-Rag1.sup.tmMom (Rag1, Jackson Lab)
mice. Six mice were used for each type of the infected cancer
cells. The longest and shortest diameters of the solid tumors were
measured using a digital caliper at the end of the experiments.
Tumor volumes were calculated using the following formula: tumor
volume=1/2.times.(shortest diameter).sup.2.times.longest diameter
(mm.sup.3). At end of the experiments, tumors were fixed and
sectioned for histological and immunohistochemical analyses.
Additional Cancer Models
[0270] Xenograft and orthotopical mesothelioma tumor models in
Rag-1 mice: 5.times.10.sup.6 of human malignant mesothelioma cells,
H-MESO-1, H-MESO-1A, and/or MSTO-211H (ATCC and NCI-DCTD Tumor/Cell
line repository in Frederick) will be injected subcutaneously and
orthotopically into the right pleural cavity immunocompromised Rag
1 mice.
[0271] Xenograft melanoma models: 5.times.10.sup.6 of human
melanoma cells, MEWO, SKMEL5, SKMEL2, and/or A375 (ATCC and
NCI-DCTD Tumor/Cell line repository in Frederick), will be injected
subcutaneously into immunocompromised Rag 1 mice.
[0272] Xenograft sarcoma models: 5.times.10.sup.6 of human sarcoma
cells, SKN-MC and A673 cell (ATCC), will be injected subcutaneously
into immunocompromised Rag 1 mice.
[0273] Xenograft pancreatic cancer models: 5.times.10.sup.6 of
human pancreatic cancer cells, Pane-1, HPAC, MIA PaCa-2, and/or
AsPC-1 pancreatic cancer cells, will be injected subcutaneously
into immunocompromised Rag 1 mice.
[0274] Xenograft hepatoma models: 5.times.10.sup.6 of human
hepatoma cells, Hep 3B2.1-7 hepatoma cells, will be injected
subcutaneously into immunocompromised Rag 1 mice.
[0275] Xenograft multiple myeloma models: 5.times.10.sup.6 of human
multiple myeloma cells, U266 and MC/CAR cells, will be injected
subcutaneously into immunocompromised Rag 1 mice.
[0276] Ascites ovarian cancer model in Rag-1 mice: 5.times.10.sup.6
of human SKOV3ip and OVCAR-3ip human ovarian cancer cells will be
injected into Rag-1 mice intraperitoneally (ip).
[0277] Xenograft and/or bone metastatic prostate cancer models:
5.times.10.sup.6 of human prostate cancer cells, 22Rv1, will be
injected into Rag-1 mice subcutaneously or intracardiacally into
each Rag-1 mice, respectively.
[0278] Xenograft and/or metastatic lung cancer models:
5.times.10.sup.6 of human lung cancer cells, A549 and LX529 will be
injected into Rag-1 mice subcutaneously or intravenously into Rag-1
mice, respectively.
[0279] Xenograft and orthotopical breast cancer models:
5.times.10.sup.6 of human breast cancer cells, MX-2 and SW613, will
be injected subcutaneously or into Rag-1 mouse mammary fat pad,
respectively.
[0280] Cancer stem cell models: Fresh human glioblastoma, human
melanoma, lung, breast, prostate, ovarian, head-neck, kidney, and
colon cancer tissues were obtained from Cooperative Human Tissue
Network (CHTN) at University of Pennsylvania and The Ohio State
University. The tissues were dissociated into single cells by 0.4%
collagenase type I (Sigma C0130) and plated in ultra-low attachment
plates in serum-free cancer stem cell culture medium, which is
DMEM/F12 supplemented with B27 (Invitrogen), EGF (10 ng/mL, BD
Biosciences), and FGF-2 (20 ng/mL, BD Biosciences). After formation
of the initial spheres, the tumor spheres were passaged
approximately every week by dissociating the spheres with 0.05%
trypsin-ethylenediamine tetraacetic acid (EDTA). The tumor spheres
were implanted subcutaneously into Rag-1 mice.
[0281] Statistics
[0282] One-way ANOVA statistic analyses were performed to analyze
statistical differences of the tumor volumes and growth rates
between the control and experimental groups. LogRank analyses were
performed for the survival experiments. Differences were considered
statistically significant at p<0.05.
Example 1
CD44 is Upregulated in Human Glioblastoma Multiforme (GBM)
[0283] To determine the expression level of CD44 in GBM, available
gene expression datasets at www.oncomine.org were mined. In four
independent datasets, CD44 transcripts were consistently
upregulated in human GBM compared to either normal brain (FIG. 1A,
studies 1, 2, and 4) (Bredel et al., 2005; Liang et al., 2005; Sun
et al., 2006) or normal white matter (FIG. 1A, study 3) (Shai et
al., 2003). Immunohistochemistry of paraffin sections of primary
tumors showed that CD44 is upregulated in all 14 GBM cases analyzed
compared to eight cases of normal human brain (FIG. 1B).
[0284] To address the role of CD44 in glioma growth and
progression, expression levels of the CD44 protein in a variety of
human glioma cell lines were analyzed. Human glioma cell lines were
derived from ATCC, UCSF, and NCI-DCTD Tumor/Cell line repository in
Frederick. The majority of human glioma cells tested express higher
levels of CD44 than normal human astrocytes (NHAs) and the standard
85-90 kDa form (CD44s, FIG. 1C) was the predominant isoform
expressed. Based on their high CD44 expression level and their
tumorigenicity in immunocompromised mice, U87MG and U251 human
glioma cells were selected to investigate the role of CD44 in
glioma growth and progression and the mechanisms whereby CD44 may
contribute to the processes.
Example 2
Lentiviral Based shRNAs Effectively Knocked Down CD44 Expression in
Human Glioblastoma Multiforme (GSM) Cells
[0285] To knock down endogenous CD44 expression effectively in U251
and U87MG cells, a set of human CD44-specific TRC-shRNA
(shRNA-TRC-CD44#1-#5) and shRNAmir (shRNAmir-CD44#1-#3) constructs
(Open Biosystems) were screened. Non-targeting control shRNAs
(shRNA-TRC-NT and shRNAmir-NT) were included in the screen as
negative controls. These shRNA vectors were lentiviral-based and
contained the internal ribosome entry site (IRES)/GFP and/or
puromycin-resistance gene located at the 3'-termini of the shRNA
inserts. The IRES element in the shRNAmir construct ensures that
all the puromycin-resistant cells express the inserted shRNAs and
allows use of the GFP expression level as an indicator of the shRNA
expression efficiency. Lentiviruses containing these shRNA
constructs were used to infect U87MG-Luc and U251-Luc cells that
had been transduced with and expressed luciferase. Luciferase
activity allowed efficient monitoring of intracranial growth of
these cells (Lau et al., 2008). After selection of the infected
cells with puromycin, expression levels of endogenous CD44 were
assessed in pooled populations of puromycin-resistant GBM cells.
Two out of three shRNAmir constructs (shRNAmir-CD44#1 and
shRNAmir-CD44#3) and 1-2 TRC-shRNA (shRNA-TRC-CD44#3 and/or
shRNA-TRC-CD44#4) knocked down CD44 expression efficiently in these
two glioma cell lines, as assessed by real-time qPCRs (data not
shown) and Western blot analysis (FIG. 2A) and immunocytochemistry
(FIG. 2B, D). Other CD44-specific shRNAs reduced CD44 expression in
variable degrees whereas the non-targeting controls displayed no
effect. Because CD44 is a major cell surface receptor of HA, the
capacity of CD44-depleted cells to bind fluorescein-labeled HA
(FL-HA) was assessed. Effective knockdown of CD44 expression
dramatically reduced the ability of glioma cells to bind and
endocytose FL-HA, whereas non-targeting shRNAs had no effect (FIG.
2C, and data not shown).
Example 3
Depletion of CD44 Expression Inhibited Subcutaneous Growth of U87MG
and U251 Cells by Inhibiting their Proliferation and Promoting
Apoptosis In Vivo
[0286] Pooled populations of the transduced U87MG and U251 cells
that displayed different degrees of CD44 depletion were first used
in subcutaneous (s.c.) tumor growth experiments to determine how
reduced CD44 expression affects glioma growth in vivo. Reduced CD44
expression in these cells correlated with reduced tumor volumes 5
weeks following injection of the GBM cells (FIG. 3A-B). Growth
curves of tumors derived from the glioma cells infected with
control non-targeting shRNAs, shRNA-TRC-NT and shRNAmir-NT, or two
CD44-specific shRNAs that effectively knock down CD44 expression,
shRNATRC-CD44#3 and shRNAmirCD44#1, further demonstrated that CD44
depletion significantly inhibited subcutaneous glioma growth (FIG.
3C-D). To begin to address the mechanisms underlying the growth
inhibitory effect of CD44 knockdown, proliferation and survival of
the transduced U87MG and U251 cells in situ were analyzed. shRNAs
that knock down CD44 expression, but not the control non-targeting
shRNAs, inhibited glioma cell proliferation (FIG. 3E-e-h) and
promoted apoptosis in vivo (FIG. 3E-i-l).
Example 4
Knockdown of CD44 Expression Inhibited Intracranial Growth of U87MG
and U251 Gliomas
[0287] To determine the effect of CD44 knockdown on intracranial
glioma growth, double drug-resistant pooled populations of glioma
cells that express high levels of luciferase and display
significant CD44 depletion were injected intracranially into
immunocompromised Rag-1 mice. Three, six, nine, and thirteen days
after injection, bioluminescence images of the intracranial tumors
were acquired using an IVIS-200 imaging system (Xenogen, FIG. 4A
and data not shown). The mice were closely monitored for the
duration of their survival as defined in Materials and Methods.
Suppression of CD44 expression significantly inhibited intracranial
tumor growth and increased the survival time of the experimental
animals compared to mice injected with U87MG/U251-Luc cells
transduced with non-targeting shRNAs cells (FIG. 4B).
[0288] To confirm the effect of reduced CD44 expression on
intracranial glioma growth, an inducible CD44 knockdown system was
established in U87MG-luc and U251-Luc cells by using two TRIPZ
lentiviral Tet-On shRNAmir constructs (Open Biosystems), which
contain two of the effective shRNAs against CD44 (shRMAmir #1 and
#3, FIG. 2 and data not shown). These TRIPZ constructs expressed
shRNAs in the presence of doxycycline (Dox) and effectively knocked
down CD44 expression in U87MG and U251 cells, whereas control
non-targeting TRIPZ shRNA had no effect on CD44 expression (data
not shown). Immunocompromised Rag-1 mice were fed regular or
doxycycline-impregnated (625 ppm; Harlan-Teklad) food pellets for
three days prior of intracranial injection of glioma cells. The
experimental mice were continuously fed with regular or
doxycycline-impregnated food pellets throughout the experiments.
Inducible knockdown of CD44 inhibited intracranial glioma growth
and prolonged mouse survival (data not shown), supporting initial
observations.
Example 5
Reduced CD44 Expression Sensitizes Glioma Cells to Cytotoxic Drugs
In Vivo
[0289] The first-line cytotoxic drugs for GBM are temozolomide
(TMZ) and carmustine (BCNU). Based on previous observations that
CD44 provides essential survival signals to metastatic breast
cancer cells (Yu et al., 1997), the possibility that reduced CD44
expression may inhibit survival signaling and sensitize glioma
cells to BCNU and TMZ treatment in vivo was addressed. Mice were
injected intracranially with U87MG-Luc and U251-Luc cells, depleted
or not of endogenous CD44, and treated sequentially with a single
dose of BCNU (10 mg/kg, iv) or TMZ (5 mg/kg, ip). BCNU and TMZ
displayed a weak and a moderate inhibitory effect on glioma growth,
respectively, when used as single agents (FIG. 4C-D). CD44
depletion, however, sensitized the response of glioma cells to BCNU
and TMZ, as demonstrated by the observation that the combination of
CD44 knockdown and treatment with BCNU or TMZ resulted in a
synergistic inhibition of intracranial glioma formation as
determined by markedly prolonged the median survival length of the
mice (FIG. 4D).
Example 6
CD44 Attenuated Activation of the Mammalian Equivalent of Hippo
Signaling Pathway and Played a Key Role in Regulating Stress and
Apoptotic Responses of Human GBM Cells
[0290] Radiation therapy provides another option for GBM patients.
Radiation therapy and some cytotoxic agents generate reactive
oxygen species (ROS), which constitute a major inducer of cell
death resulting in their anti-glioma effects. To address the
molecular mechanisms that underlie the observed chemosensitizing
effect of CD44 knockdown on glioma cells, how reduced CD44
expression affects GBM cell response to oxidative stress induced by
H.sub.2O.sub.2 and cytotoxic stress induced by TMZ was
investigated. U87MG cells transduced with a mixture of viruses
carrying the control non-targeting shRNAmir-NT and TRC-NT or with a
mixture of two of most effective shRNAs against CD44
(shRNAmirCD44#1 and TRC-CD44#3, FIG. 2) were used in these
experiments. Reduced expression of endogenous CD44 in human GBM
cells resulted in the enhanced and sustained response of the cells
to oxidative and cytotoxic stresses and reduced viability of these
cells (FIG. 5 and data not shown).
[0291] MST1/2 plays an important role in mediating
oxidative-stress-induced apoptosis (Lehtinen et al., 2006), and we
have shown that MST1/2 functions downstream of merlin in human GBM
cells (Lau et al., 2008). Compared to the GBM cells expressing a
high level of endogenous CD44, the cells with depleted endogenous
CD44 responded to oxidative stress with robust and sustained
phosphorylation/activation of MST1/2 and Lats1/2,
phosphorylation/inactivation of YAP, and reduced expression of
cIAP1/2 (FIG. 5A-B). These effects correlate with reduced
phosphorylation/inactivation of merlin, increased levels of cleaved
caspase-3 and reduced cell viability (FIGS. 5B and 3E, and data not
shown). By contrast, a higher level of endogenous CD44 promotes
phosphorylation/inactivation of merlin, inhibits the stress induced
activation of entire mammalian equivalent of Hippo signaling
pathway and up-regulated cIAP1/2, leading to inhibition of
caspase-3 cleavage and apoptosis (FIG. 5A, 3E, data not shown).
Together, these results place CD44 upstream of the mammalian Hippo
signaling pathway (merlin-MST1/2-Lats1/2-YAP-cIAP1/2) and suggest a
functional role for CD44 in attenuating tumor cell responses to
stress and stress-induced apoptosis.
[0292] Because MST1/2 kinases have multiple downstream effectors
and are implicated in several signaling pathways, whether known
effectors of MST1/2 also function downstream of this newly
established CD44-MST1/2 signaling axis was investigated. These
results indicate that knockdown of CD44 results in elevated and
sustained activation of JNK and p38 stress kinases in glioma cells
exposed to oxidative stress (FIG. 5D). In addition, oxidative
stress induced sustained up-regulation of p53, a known downstream
effector of JNK/p38, and its target genes p21 and puma in
CD44-deficient glioma cells (FIG. 5D), whereas the GBM cells with
high levels of endogenous CD44 attenuated activation of JNK/p38,
and inhibited induction of p53, p21, and puma (FIG. 5C).
[0293] Caspase-3 cleavage is an indicator of cellular apoptosis.
The in vitro data using H.sub.2O.sub.2 treatment demonstrates
caspases-3 cleavage (FIG. 5 B), suggesting that the combination of
the reduced expression of endogenous CD44 in human GBM cells with
oxidative stress would result in a decrease in GBM tumor size.
[0294] Although H.sub.2O.sub.2 was not administered in vivo,
chemotherapy and radiation therapy act to generate H.sub.2O.sub.2.
FIG. 4 (C-D) demonstrates that the combination of a reduction in
the expression of endogenous CD44 in human GBM cells coupled with
chemotherapeutic agents results in a decrease in tumor size and an
increase in survival time. Therefore, it would be expected that a
reduction in the expression of endogenous CD44 in human GBM cells
coupled with radiation therapy would act to decrease the GBM tumor
size as well as increase the survival time as both types of
therapies would result in the production of H.sub.2O.sub.2.
[0295] To address the mechanism whereby CD44 depletion sensitizes
glioma cells to cytotoxic drugs in vivo (FIG. 4C-D), similar
experiments were performed to those outlined in FIG. 5 but using
TMZ instead of H.sub.2O.sub.2 to induce cytotoxic stress in the
glioma cells with high or low CD44 expression. Similar to their
response to oxidative stress, glioma cells expressing a very low
level of CD44 mounted robust and sustained activation of MST1/2
upon exposure to TMZ, along with phosphorylation/inactivation of
YAP that correlated with reduced levels of cIAPs, activation of p38
but not JNK, and up-regulation p53 and its target gene p21 (FIG.
6). Together, these results establish a novel role of CD44 in
inhibiting stress/apoptotic responses of tumor cells by attenuating
activation of the mammalian Hippo signaling pathway and provide a
first molecular explanation for how up-regulation of CD44 may
constitute a key event in tumor cell resistance to stress of a
broad range of origins, including that generated by host defense
and therapeutic intervention.
Example 7
CD44 Modulated ErbB and c-Met Receptor Tyrosine Kinase (RTK)
Mediated Growth-Signaling Pathways in Glioma Cells
[0296] In vivo results show that CD44 knockdown inhibits
proliferation of the GBM cells in vivo (FIG. 3E). Previous studies
have shown that CD44 is a co-stimulator of ErbB and c-Met RTK
signaling pathways (Orian-Rousseau et al., 2002; Toole, 2004; van
der Voort et al., 1999), which may account for the reduced in vivo
proliferation of CD44-depleted glioma cells, given that RTK
signaling pathways are strongly implicated in glioma progression.
To determine whether knockdown of CD44 diminishes EGF family
ligand- and HGF-induced activation of the downstream signaling
pathways, serum starved CD44-high or -low U87MG cells were treated
with different RTK ligands, including EGF family ligands,
heparin-binding EGF (HB-EGF), betacellulin (BTC), amphiregulin (AR)
and epiregulin (Epr)), HGF, NGF, and 10% fetal bovine serum (FBS).
Reduced expression of CD44 diminished EGF family ligand- and HGF-
but not NGF- and FBS-induced phosphorylation of Erk1/2 kinase but
not that of AKT kinase (FIG. 7), suggesting that CD44
preferentially modulates proliferation but not survival signaling
pathways activated by these growth factors and that CD44 regulates
survival signaling pathway through the Hippo pathway.
Example 8
Transcript Profiling of U87MGmerlin and WM793merlin Cells Suggests
that Merlin, a Downstream CD44 Effector that is Negatively
Regulated by CD44, is a Mediator of a Master Regulator of Several
Important Signaling Pathways
[0297] CD44 and merlin negatively regulate each other function (Bai
et al., 2007 and Xu et al., 2010). U87MG cells responded to the
growth inhibitory effect of merlin in a dramatic fashion (Lau et
al., 2008), suggesting that downstream signaling pathways of merlin
are intact in these cells even though merlin expression is down
regulated and CD44 expression is up-regulated. This cell model
results in an excellent opportunity to identify the differentially
expressed genes and the altered signaling pathways in response to
merlin re-expression. These differentially expressed gene may
represent the essential downstream effectors of merlin and CD44,
which are likely either hyperactive or hypoactive when merlin
function is lost or impaired and CD44 is up-regulated in human
gliomas. Deregulation of these signaling pathways may lead to
gliomagenesis and/or devastating progression of this disease.
[0298] To identify downstream effectors that mediate the potent
anti-glioma effect of merlin, gene expression profiles of three
independently-transduced and pooled U87MG.sub.merlin and
U87MG.sub.wt cells, which express high and low level of merlin,
respectively, were compared using human U133v2 gene chips
(Affymetrix). The microarray results indicated that the expression
of merlin in U87MG.sub.merlin cells is .about.three fold higher
than in U87MG.sub.wt cells. 362 genes whose expression increased
and 364 genes whose expression decreased in U87MG.sub.merlin cells
compared to U87MG.sub.wt cells were identified. They can be
categorized into the genes involved in adhesion, migration,
organization of actin-cytoskeleton, cell cycle, survival, and
signal transduction. These genes were imported to David Functional
Annotation Bioinformatics Microarray Analysis software
(http://david.abcc.ncifcrf.gov/home.jsp, NIAID/NIH) to enrich for
functionally related gene groups. After classification of these
transcripts into functional pathways, we found that merlin
re-expression results in increased expression of transcripts that
activates Hippo signaling pathway as well as increased expression
of molecules that inhibit Wnt signaling pathway and decreased
expression of transcripts that activate Wnt and HGF/c-Met and
pleiotrophin (PTN)/Anaplastic lymphoma kinase (ALK) signaling
pathways (FIG. 8).
[0299] To establish the common changes in the expression profiles
induced by merlin among different tumor types, the effect of merlin
on human melanoma growth was investigated. It was determined that
merlin expression is down-regulated in human melanoma cell lines
and that increased expression of wt merlin significantly inhibits
subcutaneous growth of WM793 human melanoma cells in vivo (data not
shown). Further assessment of the transcript profiles of
WM793.sub.wt and WM793.sub.merlin cells demonstrated that increased
expression of merlin significantly up-regulates 697 genes, many of
which display anti-tumor properties, and down-regulates 736 genes,
many of which display pro-tumor activity (data not shown). These
significantly up- and down-regulated genes were imported to David
Functional Annotation Bioinformatics Microarray Analysis software
to enrich functional-related genes and generate the signaling
pathways that are significantly affected by increased expression of
merlin. These outputted data were then compared with that derived
from U87MG glioma cells and the common alterations induced by
merlin were identified. Together, these data indicated that
increased expression of merlin activates Hippo and inhibits Wnt and
c-Met signaling pathways (FIG. 8).
Example 9
Merlin Inhibits Wnt-Signaling
[0300] These merlin-induced changes of expression were then
investigated at the functional level. Since canonical Wnt signaling
regulates gene expression by modulating the levels of beta-catenin
expression, a co-activator of the T-cell factor/lymphocyte enhancer
factor (TCF/LEF) transcription factors, reporter assays using a
beta-catenin-responsive luciferase reporter construct, TopFlash
(Addgene), were performed. FopFlash, which contains mutated TCF/LEF
binding sites, was used as a negative control. It was found that
beta-catenin transcriptional activity is inhibited by wild-type
merlin and merlinS518A, but not by merlinS518D (FIG. 9) (Lau et
al., 2008).
Example 10
CD44 and Merlin-Mediated Signaling Events and their Potential
Cross-Talk
[0301] A working model of CD44 and merlin-mediated signaling events
and their potential cross-talk (the components of Drosophila Hippo
signaling pathway are underlined): merlin functions upstream of the
mammalian Hippo (merlin-MST1/2-LATS1/2-YAP) and JNK/p38 signaling
pathways and plays an essential role in regulating the cell
response to the stresses and stress-induced apoptosis as well as to
proliferation/survival signals. Merlin antagonizes CD44 function
and inhibits activities of RTKs and the RTK-derived growth and
survival signals. CD44 function upstream of mammalian Hippo
signaling pathway and enhances activities of RTKs
Example 11
Antagonists of CD44, hsCD44-Fc Fusion Proteins, Serve as Effective
Therapeutic Agents Against Human GBM in Mouse Models
[0302] To determine whether antagonists of CD44 can be used to
inhibit glioma progression in preclinical mouse models, several
fusion proteins composed of the constant region of human IgG1 (Fc)
(Holash et al., 2002; Kim et al., 2002; Sy et al., 1992) fused to
the extracellular domain of CD44v3-v10, CD44v8-v10, CD44s,
CD44v3-v10R41A, CD44v8-v10R41A, or CD44sR41A were developed (FIG.
11). The antibody-like characteristics of these fusion proteins
provide them with favorable pharmacokinetics and biodistribution
profile in vivo in addition to relative ease of production and
purification in vitro. Receptor-Fc fusion proteins may function by
trapping ligands and/or by interfering with endogenous receptor
functions.
[0303] Mutating R41 to A abolishes the ability of CD44 to bind to
HA. The ability of the mutated CD44 to bind to all other ligands
and CD44 sheddases, however, will likely be preserved, which is
important because ligands other than HA and the CD44 sheddase are
likely to be very important to exert the pro-tumor activity of
CD44. While the loss of HA binding may reduce some activity of
CD44R41A-Fc against certain cancers, this modification may improve
the biodistribution and bioavailability.
[0304] The v3 exon of CD44 contains a Ser-Gly-Ser-Gly motif for
covalent attachment of heparan sulfate (HS) side chains (Bennett et
al., 1995). To assess whether hsCD44v3-v10-Fc proteins are modified
by HS, purified hsCD44s-Fc, hsCD44v8-v10-Fc, and hsCD44v3-v10-Fc
fusion proteins were treated with or without heparinase I/III
before elution from protein A columns. These proteins were then
coated on Elisa plates in triplicate. After blocking with BSA, the
coated proteins were tested for reactivity with anti-HS antibody.
The intensity of the reaction, as assessed by a colorimetric assay,
was normalized by the reactivity with anti-CD44 antibody, which
provides relative quantity of the coated fusion proteins on the
plates. These results showed that only hsCD44v3-v10-Fc was modified
by HS and stained positively with anti-HS antibody. The observed
reactivity was sensitive to heparinase I/III treatment (FIG.
11C).
[0305] U87MG and U251 cells were transduced with retroviruses
carrying the expression constructs encoding these CD44-Fc and
CD44R41A-Fc fusion proteins or empty expression vector. Pooled
puromycin resistance cells expressed high levels of
hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, hsCD44s-Fc, hsCD44v3-v10R41A-Fc,
hsCD44v8-v10R41A-Fc, hsCD44sR41A-Fc fusion proteins (FIG. 11A,FIG.
12A). Whether the soluble CD44-Fc fusion proteins are capable of
altering FL-HA binding to endogenous GBM cell surface CD44 was
assessed. It was found that expression of the CD44-Fc fusion
proteins reduced binding of FL-HA to the GBM cells (FIG. 11B).
These cells were then compared to empty vector-transfected cells
for subcutaneous and intracranial growth in Rag-1 mice.
hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc expression
markedly inhibited subcutaneous and intracranial growth of U87MG
and U251 cells and significantly extended survival of mice bearing
the intracranial tumors. The hsCD44v3-v10-Fc fusion protein
displayed the most profound inhibitory effect (FIGS. 12B and
C).
[0306] CD44 has multiple ligands including HA, osteopontin, heparin
binding growth factors, fibronectin, serglycin, laminin, MMP-9, and
fibrin (Bennett et al., 1995; Ponta et al., 2003; Stamenkovic,
2000; Stamenkovic and Yu, 2009; Toole, 2004) and cooperates with
several RTKs and other cell surface receptors (Orian-Rousseau et
al., 2002; Stamenkovic, 2000; Stamenkovic and Yu, 2009). Many of
CD44 functions are mediated through its interaction with HA (Toole,
2004), which is abolished by the single R41A mutation (Peach et
al., 1993). To determine whether the CD44-HA interaction alone is
responsible for the GBM promoting activity of CD44, pooled
populations of U87MG and U251 cells expressing hsCD44sR41A-Fc or
hsCD44v3-v10R41A-Fc were generated and their anti-GBM effects were
compared with that of their wild type counterparts. Unlike wild
type CD44-Fc fusion proteins, CD44R41A-Fc proteins are incapable of
inhibiting FL-HA binding to the GBM cells (FIG. 11B and data not
shown). However, whereas hsCD44sR41A-Fc displayed a weak anti-GBM
effect, hsCD44v3-v10R41A-Fc retained a substantial level of
anti-GBM activity (FIG. 12C-c and data not shown), which is
consistent with the finding that hsCD44v3-v10-Fc fusion protein
exerts the most potent anti-GBM effect of the three CD44-Fc fusion
proteins tested, suggesting a mechanism of action in addition to
trapping HA. Together, these results suggest that CD44, and
especially CD44 variants, promote tumor progression both in an
HA-dependent and HA-independent fashion.
[0307] Finally, the anti-GBM efficacy of purified hsCD44s-Fc fusion
proteins in pre-established intracranial gliomas resulting from
injection of 5.times.10.sup.5 U87MG or U251 cells into Rag-1 mice
was assessed. Intracranial tumors were grown for 5 days before the
mice were treated by intravenous injection of 0.9% NaCl containing
5 mg/kg human IgG or purified hsCD44s-Fc fusion proteins every
third day until completion of the experiments. Systemic delivery of
hsCD44s-Fc fusion proteins but not human IgG markedly inhibited
intracranial growth of U87MG and U251 cells and significantly
(p<0.001) extended median survival of the experimental mice
(FIG. 13 A-B). GBM and brain issue were collected at the time of
mouse euthanasia, sectioned, and stained with anti-human IgG
antibody to assess the bio-distribution of hsCD44-Fc fusion
proteins. The results showed that hsCD44-Fc fusion proteins readily
penetrated tumor blood vessels and displayed a remarkable
intra-glioma distribution pattern whereas negligible fusion
proteins were observed in normal adjacent brain tissue, most likely
due to the presence of an intact blood-brain-barrier (FIG. 13C).
These results show that CD44-Fc proteins preferentially accumulate
within the tumor tissue, which contains leaky blood vessels.
Together, the results demonstrate that hsCD44-Fc fusion proteins
are potentially attractive therapeutic agents for GBM. In addition,
normal host tissues were stained with H&E to assess potential
toxicity of systematical delivery of the fusion proteins. Upon
carefully gross and histological examination, no apparent toxicity
and necrosis to normal tissues were observed (FIG. 13D).
Example 12
Knockdown of CD44 Sensitizes the Responses of Cancer Cells to the
erbB and c-Met RTK Inhibitors
[0308] As shown in FIG. 7, CD44 plays an important role in
enhancing the growth signals derived from ErbB and c-Met RTKs. To
determine whether knockdown of CD44 sensitizes the responses of GBM
cells to the pharmacologic inhibitors of erbB and c-Met RTKs,
glioma cell viability assays in the presence or absence of
different concentrations of inhibitors of erbB and c-Met RTKs, with
or without CD44 knockdown, were performed. The results showed that
shRNAs knocked down CD44 expression sensitized the response of
U87MG cells to a dual inhibitor of EGFR/erbB-2 (BIBW2992), a pan
inhibitor of EGFR/erbB2/4 (CI-1033) and a c-Met inhibitor (SU11274;
LC Laboratories, Selleck Chemicals Co.) (FIG. 14), providing
evidence that targeting CD44 and erbB or c-Met together can achieve
synergistic inhibitory effects in the cancer where these molecules
play important roles.
Example 13
hsCD44-Fc Fusion Proteins Sensitize the Responses of GBM Cells to
Chemotherapy and Targeted Therapy
[0309] To determine whether CD44 antagonists, hsCD44s-Fc fusion
proteins, sensitize the responses of GBM cells to chemotherapeutic
agents and pharmacologic inhibitors of erbB and c-Met RTKs, glioma
cell viability assays in the presence or absence of different
concentrations of TMZ, inhibitors of erbB and c-Met RTKs with or
without purified hsCD44s-Fc fusion proteins or human IgG were
performed. The results showed that hsCD44s-Fc fusion proteins but
not human IgG sensitize the response of U87MG cells to TMZ, a dual
inhibitor of EGFR/erbB-2 (BIBW2992), a pan inhibitor of
EGFR/erbB2/4 (CI-1033), and a c-Met inhibitor (PF-2341066, Selleck
Chemicals Co.) (FIG. 15).
Example 14
hsCD44s-Fc Fusion Proteins Display Low Cytotoxicity Towards a Panel
of Normal Cells
[0310] Two important characteristics used to define good cancer
therapy targets are high expression of the targets in tumor cells
and low or absent expression in normal cells and increased
dependency of tumor cells on the target functions. CD44 meets these
criteria. To assess potential toxicity of CD44-Fc fusion proteins
towards normal cells, cell viability assays using a panel of normal
cells in the presence or absence of different amount of purified
hsCD44s-Fc fusion proteins were performed. The results demonstrated
that CD44-Fc fusion proteins displayed low toxicity towards normal
human astrocytes (NHAs), Schwann cells, fibroblasts (HGF-1) and
endothelial cells (HUVECs) comparing to U251 GBM cells (FIG.
16).
Example 15
CD44 is Required for Self-Renewal and In Vivo Growth of GBMCSCs
[0311] Stem cells exhibit the characteristic of self-renewal. To
determine the contribution of CD44 to the self-renewal capacity of
glioma CSC spheres, primary human glioma spheres (HGSs) from fresh
GBM tissues (CHTN) were established. Human GBMCSC spheres,
MSSM-GBMCSC-1 and -2, derived from fresh GBM tissues have
self-renewal capacity, express stem cell markers (Sox-2 and
nestin), and can be readily transduced using retro- and
lenti-viruses to express or to knock down expression of the genes
of interests (FIG. 17A-C). shRNAs knocked down of CD44 expression
in theses GBMCSCs inhibited the sphere formation (FIG. 18),
demonstrating that CD44 is important for maintenance glioma stem
cells and its targeting helps to eliminate cancer stem cells and
stop the recurrence of malignant cancers. In addition, it was found
that MSSM-GBMCSC-1 and -2 readily form invasive intracranial tumors
in Rag-1 mice (FIG. 17D and data not shown) and overexpression of
hsCD44s-Fc fusion proteins inhibits intracranial growth of
MSSM-GBMCSC-1 cells (FIG. 17D-b).
Example 16
CD44 is Up-Regulated in a Variety of Human Cancer Types
[0312] To determine the expression level of CD44 in colon cancer,
ovarian cancer, head and neck squamous carcinoma, renal cell
carcinoma, melanoma, gastric cancer, and esophageal cancer,
available gene expression datasets at www.oncomine.org were mined.
We found that CD44 transcripts were up regulated in human colon
(FIG. 19A, 19C) (Graudens et al., 2006; Notterman et al., 2001),
ovarian (FIG. 32A) (Hendrix et al., 2006), head and neck squamous
carcinoma (FIG. 38A, FIG. 39) (Ginos et al., 2004), renal cell
carcinoma (FIG. 37, FIG. 38B) (Gumz et al., 2007), melanoma (FIG.
35A-B), gastric cancer (FIG. 42), and esophageal cancer (FIG. 43)
compared to their normal counterparts. Data was derived from
oncomine (www.oncomine.org).
[0313] In addition, immunohistochemistry analysis of paraffin
sections of primary human tumors showed that CD44 is up regulated
in malignant/metastatic colon cancer (FIG. 19B), prostate cancer
(FIG. 22), malignant breast cancer (FIG. 25), and metastatic
ovarian cancer (FIG. 32B-C) comparing to their normal counterpart
tissues or primary tumors.
Example 17
Knockdown of CD44 Expression Inhibited the In Vivo Growth of a
Variety of Human Cancer Cells
[0314] To knock down endogenous CD44 expression in HCT116 and
KM20L2 human colon cancer cells, PC3/M human prostate cancer cells,
MX-2 and SW613 human breast cancer cells, NCI-H125 and NCI-H460
human lung cancer cells, and OVCAR-3 human ovarian cancer cells, a
set of human CD44-specific TRC-shRNA (shRNA-TRC-CD44#1-#5) and
shRNAmir (shRNAmir-CD44#1-#3) constructs (Open Biosystems) were
screened. Non-targeting control shRNAs (shRNA-TRC-NT and
shRNAmir-NT) were included in the screen as negative controls.
Lenti-viruses containing these shRNA constructs were used to infect
the cancer cells. Following selection of the infected cells with
puromycin, the expression level of endogenous CD44 was assessed in
pooled populations of puromycin-resistant cancer cells. At least
two shRNAs effectively knocked down CD44 expression in these cancer
cells as assessed by western blot analysis (FIG. 20A, 21A, 23A,
26A, 27A, 30-31A, and 33A). Other CD44-specific shRNAs reduced CD44
expression in variable degrees, whereas the non-targeting controls
displayed no effect. Pooled populations of these transduced cancer
cells, displaying different degrees of CD44 depletion, were used in
subcutaneous (s.c.) tumor growth experiments to determine how
reduced CD44 expression affects their subcutaneous growth in vivo.
Results showed that reduced CD44 expression in these cells
correlated with reduced tumor volumes 6 weeks following injection
of these cells (FIG. 20-21B, 23B, 26-27B, 30-31B, and 33B),
establishing that CD44 is required for in vivo growth of these
types of cancer cells, and therefore, is a prime target of
therapeutic intervention of these cancer types.
Example 18
Purified CD44-Fc Fusion Proteins Inhibit In Vivo Growth of Human
Prostate Cancer
[0315] CD44 expression by three human prostate cancer cell lines
was assessed. It was found that the most aggressive prostate cell
line, PC3/M cell, expresses the highest level of CD44 (FIG. 24A).
To assess the effect of purified hsCD44-Fc fusion proteins on PC3/M
cell growth in vivo, 5.times.10.sup.6 PC3/M cells were injected
subcutaneously into each Rag-1 mice. The tumors were allowed to
growth for .about.two weeks when the tumor volumes reach .about.150
mm.sup.3. The mice bearing similar size tumors were separated into
6 groups (6mice/group) and were treated with 4 intratumoral
injections of 5 .mu.l/injection of 10 mg/ml of hsCD44s-Fc,
hsCD44v8-v10-Fc, hsCD44v6-v10-Fc, hsCD44v3-v10-Fc, or human IgG, or
0.9% NaCl (FIG. 24B). The experiments were stopped when the tumors
of the control groups (treatment of human IgG or 0.9% NaCl) reached
1 cm in their longest diameters. All the tumors were dissected out
and weighted. Our results showed that CD44-Fc fusion proteins but
not 0.9% NaCl or human IgG significantly inhibited growth of PC3/M
cells in vivo (FIG. 24B).
Example 19
Human Malignant Breast-Cancer-Cell-Infiltrated Host Stroma
Expresses a High Level of CD44 and Invasive Breast Cancer Stroma
Accumulates a Higher Level of HA
[0316] To determine the role of CD44 in breast cancer progression
and in maintenance of breast cancer stem cell (BCSC), CD44 protein
and HA levels in human malignant breast cancer tissues (obtained
from CHTN--at University of Pennsylvania) were measured. Compared
to normal breast tissues (FIG. 25A), CD44 is highly up-regulated in
the breast cancer cells infiltrated into host stroma (FIG. 25B-C).
Additionally, HA accumulates in malignant breast cancer stroma
(FIG. 25E) compared to normal breast stroma (FIG. 25D). HA in the
paraffin sections was detected by biotinylated CD44-Fc fusion
proteins.
Example 20
Establishment of Human BCSCs and In Vivo Breast Cancer Model;
Demonstrating that CD44 is Required for BCSC Self-Renewal and
Maintenance and for BCSC Growth In Vivo
[0317] Studies have shown that mammospheres are enriched for
tumorigenic BCSCs (Al-Hajj et al., 2003; Reya et al., 2001). Three
different preparations of primary mammospheres (MSSM-BCSC-1, -2,
and -3) derived from fresh malignant human breast cancer tissues
were established. These MSSM-BCSCs express high levels of the
cancer stem cells marker, CD44, and low levels of CD24 (FIG. 28).
They also express stem cell markers, Sox-2, Oct3/4, Nanog, and/or
SSEA-1 (FIG. 28B and not shown), and display self-renewal capacity
in the mammosphere formation assays (FIG. 28C-a-c) and
tumorigenicity when implanted in immunocompromised Rag-1 (FIG. 28
E). As shown in FIG. 28A, several CD44 isoforms as well as the
standard form of CD44 (CD44s, the lower band) are expressed by
MSSM-BCSCs. We established a protocol to transduce BCSCs using
retro- and lenti-viruses to express or to knock down expression of
the genes of interests. These MSSM-BCSCs were transduced with the
retroviruses carrying luciferase. After selection with G418, the
drug resistant pooled populations of MSSM-BCSC-Luc cells express
high levels of luciferase, which allowed tracking of their growth
in vivo (FIG. 28E). Furthermore, it was found that shRNAs targeting
CD44 expression inhibited mammosphere formation, while
non-targeting shRNAs had no effect on mammosphere formation (FIG.
28C-d-f, D). This results demonstrates that CD44 is important for
BCSC self-renewal and maintenance and that its target and
dysfunction may help eliminate breast cancer stem cells and
recurrence of the malignant disease.
Example 21
Purified CD44-Fc Fusion Proteins Inhibit In Vivo Growth of
BCSCs
[0318] To assess the effect of purified hsCD44-Fc fusion proteins
on BCSC growth in vivo, 1.times.10.sup.6 MSSM-BCSC-1 cells were
injected subcutaneously into each Rag-1 mice. The tumors were
allowed to growth for three weeks when the tumor volumes reach
.about.200 mm.sup.3. The mice bearing similar size tumors were
separated into 6 groups (6mice/group) and were treated with 4
intratumoral injections of 4 d/injection of 10 mg/ml of hsCD44s-Fc,
hsCD44v8-v10-Fc, hsCD44v6-v10-Fc, hsCD44v3-v10-Fc, or human IgG, or
0.9% NaCl (FIG. 29). The experiments were stopped when the tumors
of the control groups (treatment of human IgG or 0.9% NaCl) reached
1 cm in their longest diameters. All the tumors were dissected out
and weighted. The results showed that CD44-Fc fusion proteins but
not 0.9% NaCl or human IgG significantly inhibited growth of BCSCs
in vivo (FIG. 29).
Example 22
CD44 is Up Regulated in the Stroma of Human Ovarian Cancer
[0319] To determine the contribution of CD44 to the progression of
human ovarian cancer, available datasets at www.oncomine.org were
mined and it was found that the CD44 transcript is up-regulated in
human ovarian cancer comparing to normal ovary (FIG. 32A).
Immunohistochemistry analyses indicated that CD44 and its ligand,
HA, are up-regulated in the infiltrating stroma of stage III and IV
of human ovarian cancers when compared to normal ovary (FIG. 32B-D
and data not shown).
Example 23
CD44 is Important for Ovarian Cancer Stem Cell (OCSC) Self-Renewal
and Maintenance
[0320] A series of in vivo selections by intraperitoneal (ip)
implantation of parental SKOV3 and OVCAR-3 cells into Rag-1
immunocompromised mice to establish ascites ovarian cancer models
were performed. SKOV3ip and OVCAR-3ip cells derived from these
selections form subcutaneous as well as ascites tumors in Rag-1
mice (FIG. 34A and data not shown). In addition, CD44+ OCSC spheres
(MSSM-OCSC-1 and -2) from fresh metastatic ovarian cancer tissues
were generated. These MSSM-CSC cells express high levels of the
cancer stem cells marker, CD44, and they also express the stem cell
markers Sox-2, Oct3/4, and Nanog (FIG. 34B), and display
self-renewal capacity in the sphere formation assays (FIG. 34E-a-c)
and tumorigenicity when implanted in Rag-1 mice (FIG. 34C and not
shown). We established a protocol to transduce OCSCs efficiently
using retro- and lenti-viruses to express or to knock down
expression of the genes of interests. It was found that shRNAs that
knocked down CD44 expression (FIG. 34D), but not non-targeting
shRNAs, inhibited sphere formation (FIG. 34E-d-f, 34F),
demonstrating that CD44 is important for OCSC self-renewal and
maintenance and its target may lead to eliminate ovarian cancer
stem cells and stop disease recurrence.
Example 24
CD44 is Up-Regulated in Human Melanoma
[0321] To determine the contribution of CD44 to the progression of
human melanoma, available datasets at www.oncomine.org were mined
and it was found that the CD44 transcript is up-regulated in human
melanoma comparing to normal skin (FIG. 35B). Western blot analysis
also indicated that CD44 is up-regulated in human malignant
melanoma cells when compared to normal melanocytes (FIG. 35C).
Example 25
hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc Inhibit
Subcutaneous Growth of Human Melanoma Cells In Vivo
[0322] To assess the effects of expression of hsCD44-Fc fusion
proteins on melanoma growth in vivo, 2.times.10.sup.6 M14 cells
expressing different CD44-Fc fusion proteins (hsCD44s-Fc,
hsCD44v8-v10-Fc, or hsCD44v3-v10-Fc) or transduced with empty
expression vectors were injected subcutaneously into each Rag-1
mice. Tumors were allowed to grow for .about.4 weeks. At the end of
experiments, all the tumors were dissected out and weighted. Data
is presented as the mean of tumor weight (gram)+/-SD. The results
showed that CD44-Fc fusion proteins especially hsCD44v3-v10-Fc
significantly inhibited growth of M14 melanoma cells in vivo (FIG.
36B).
Example 26
CD44 is Up Regulated in Human Head-Neck Cancer
[0323] To determine the contribution of CD44 to the progression of
human head-neck cancer, available datasets at www.oncomine.org were
mined and it was found that the CD44 transcript is up-regulated in
human head-neck cancer comparing to their normal counterparts (FIG.
38A, FIG. 39). Furthermore, CD44 expression in human head and neck
carcinoma cells was assessed by Western blotting using anti-CD44
antibody (Santa Cruz). Expression level of CD44 by these carcinoma
cells correlates with their tumorigenicity in vivo (FIG. 40A).
Example 27
hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc Inhibits
Subcutaneous Growth of Human Head-Neck Cancer Cells In Vivo
[0324] To assess the effects of expression of hsCD44-Fc fusion
proteins on head-neck cancer cell growth in vivo, 5.times.10.sup.6
SCC-4 cells expressing different CD44-Fc fusion proteins
(hsCD44s-Fc, hsCD44v8-v10-Fc, or hsCD44v3-v10-Fc) or transduced
with empty expression vectors were injected subcutaneously into
each Rag-1 mice. Tumors were allowed to grow for .about.2 months.
At the end of experiments, all the tumors were dissected out and
weighted. Data is presented as the mean of tumor weight
(gram)+/-SD. The results showed that CD44-Fc fusion proteins
especially hsCD44v3-v10-Fc and hsCD44v8-v10-Fc significantly
inhibited growth of SCC-4 cells in vivo (FIG. 40C).
Example 28
hsCD44v3-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc Inhibits
Subcutaneous Growth of Human Pancreatic and Liver Cancer Cells In
Vivo
[0325] CD44 expression in human pancreatic and liver carcinoma
cells was assessed by Western blotting using anti-CD44 antibody
(Santa Cruz). The results showed that BXPC-3, PAN-08-13, PAN-08-27,
PAN-10-05 pancreatic cancer cells and SK-HEP-1 liver cancer cells
expression several CD44 isoforms (FIG. 41A).
[0326] To assess the effects of expression of hsCD44-Fc fusion
proteins on in vivo growth of pancreatic and liver cancer cells,
5.times.10.sup.6 BXPC-3 and SK-HEP-1 cells expressing different
CD44-Fc fusion proteins (hsCD44s-Fc, hsCD44v8-v10-Fc, or
hsCD44v3-v10-Fc) or transduced with empty expression vectors were
injected subcutaneously into each Rag-1 mice. Tumors were allowed
to grow for .about.5 weeks. At the end of experiments, all the
tumors were dissected out and weighted. Data is presented as the
mean of tumor weight (gram)+/-SD. The results showed that CD44-Fc
fusion proteins significantly inhibited in vivo growth of BXPC-3
and SK-HEP-1 (FIG. 41C-D).
Example 29
Methods of Detecting HA Using Biotin-Labeled CD44-Fc Fusion
Proteins and Methods of Diagnosing Cancers by Detecting HA
[0327] Purified CD44-Fc fusion proteins (hsCD44s-Fc,
hsCD44v8-v10-Fc, and hsCD44v3-v10-Fc) were labeled with biotin
using EZ-Link Biotinylation Kits (Thermo Scientific) following the
manufacturer's instruction. Human tumor paraffin sections were
deparaffinized and rehydrated. After blocking with 2% BSA, the
sections were incubated with biotinylated CD44-Fc fusion proteins
(bCD44-Fc, 1 .mu.g/ml) for overnight at 4 degree. Biotinylated
CD44-Fc fusion proteins were detected by VECTASTAIN ABC kit. Our
results showed that HA is up-regulated in stroma of malignant
breast cancer and metastatic ovarian cancer (FIG. 25E, FIG. 32D)
when compared to stroma of normal breast or ovarian (FIG. 25 D and
data not shown). The bCD44-Fc positive staining is specific for HA
as pre-treatment of the tissue sections with hyaluronidase
eliminates the staining (data not shown).
[0328] It has been well established that HA is up-regulated in many
cancer types including breast, ovarian, gladder cancer, and
prostate cancers [for review see (Simpson and Lokeshwar, 2008;
Tammi et al., 2008; Toole, 2004)](Golshani et al., 2008). HA level
is correlated to tumor progression and metastasis (Toole and
Hascall, 2002). Increased HA correlates with poor prognosis,
disease progression, and shortened overall and disease specific
survival in gastrointestinal tract, breast and ovary carcinoma
(Anttila et al., 2000; Tammi et al., 2008). A study has shown that
urinary HA measurement is an accurate marker for diagnosing bladder
cancer (Lokeshwar et al., 2000).
[0329] To detect plasma HA level, 200 .mu.l blood from each
transgenic mice (MMTV-PyVT and MMTV-ActErbb2, Jackson Lab) bearing
breast cancer, each Rag-1 mice bearing gliomas derived from
MSSM-GBMCSC-1 or Glioma 261 cells, or each control health mice were
collected. Blood samples from six mouse of each type were collected
and plasmas were generated immediately. 50 .mu.l plasma from each
sample was loaded in triplicate into each well of an Elisa plate
that has been pre-coated with CD44-Fc fusion proteins. The CD44-Fc
bound HA was detected by biotinylated CD44-Fc fusion proteins and
AP-conjugated avidin. The developed color was measure by an Elisa
machine at 405 nm. The results showed that HA is up-regulated in
the plasma samples derived from mice bearing tumors when compared
to the health mice (FIG. 44), demonstrating biotinylated CD44-Fc
fusion proteins can be used to detect HA levels in plasma, serum,
and urine of cancer patients and serve as diagnostic and prognostic
reagent.
Example 30
Reduced CD44 Expression Sensitizes Prostate Cells to Cytotoxic
Drugs In Vivo
[0330] The first-line and second-line cytotoxic drugs for prostate
cancer are docetaxel, mitoxantrone, satraplatin, and ixabepilone.
Mice will be injected subcutaneously with PC3/M-Luc and 22Rv1-Luc
cells, depleted or not of endogenous CD44, and will be treated
sequentially with docetaxel or mitoxantrone.
Example 31
Antagonists of CD44-hsCD44v3-v10-Fc, hsCD44v6-v10-Fc,
hsCD44v8-v10-Fc, and hsCD44s-Fc-Serve as Effective Therapeutic
Agents Against Various Cancers in Mouse Models
[0331] To determine whether antagonists of CD44 can be used to
inhibit progression of a variety of human cancers in preclinical
mouse models, soluble CD44 fusion proteins, such as CD44v3-v10-Fc,
CD44v6-v10-Fc, CD44v8-v10-Fc, CD44v6-v10-Fc, or CD44s will be
tested in different cancer mouse models.
[0332] These CD44-Fc fusion cDNAs have been inserted into
retroviral vectors (Clontech) that contain the IRES element
positioned between the cDNA inserts and the puromycin-resistance
gene, so that all the puromycin-resistant cells are expected to
express the inserted fusion genes. Human cancer cells, MEWO and
A375 human melanoma cells; Lovo human colon cancer cells; Panc-1,
HPAC, MIA PaCa-2, and/or AsPC-1 human pancreatic cancer cells; Hep
3B2.1-7 human hepatoma cells; SCC, -9, -15, and/or -25, human head
and neck squamous carcinoma cells; U266 and MC/CAR human multiple
myeloma cells; SKOV3ip and OVCAR -3ip human ovarian cancer cells;
and 22Rv1 human prostate cancer cells; A549, LX529, NCI-H460,
and/or NCI-H125 human lung cancer cells; MX-2 and/or SW613 human
breast cancer cells; SKN-MC and A673 human sarcoma; H-MESO-1,
H-MESO-1A, or MSTO-211H human malignant mesothelioma cells; and/or
human cancer stem cells of different origins, will be transduced
with retroviruses carrying the expression constructs encoding these
fusion proteins or empty expression vector. After selection of the
infected cells with puromycin, the pooled drug-resistant cancer
cells will express high levels of CD44 fusion proteins, such as
hsCD44v3-v10-Fc, hsCD44v6-v10-Fc, hsCD44v8-v10-Fc, and hsCD44s-Fc.
These cells will be used to assess their ability to grow in Rag-1
mice and their response to chemotherapy and other targeted
therapies.
[0333] Additional in vitro tumor cell viability experiments will be
performed using CD44 depletion and/or hsCD44-Fc fusion proteins
alone or in combination with the chemotherapeutic agent/RTK
inhibitors/IAP inhibitors/p53 activator to determine whether CD44
antagonists sensitize the response of a variety of tumor cells to
chemotherapy and other targeted therapies.
DISCUSSION
[0334] In summary, the present Examples and figures demonstrate
that CD44 is up-regulated in several human cancer types including
human glioblastoma, colon cancer, ovarian cancer, head and neck
squamous carcinoma, renal cell carcinoma, breast cancer, prostate
cancer, gastric cancer, melanoma, and esophageal cancer. CD44
antagonists including shRNAs against human CD44 and/or a variety of
CD44-Fc fusion proteins inhibit in vivo growth of human
glioblastoma, colon, breast, prostate, lung, melanoma, pancreatic
cancer, liver cancer, head and neck carcinoma, pancreatic, and
ovarian cancers in mouse models. Moreover, the Examples demonstrate
that CD44 is upregulated in human GBM and that knockdown of CD44
inhibits GBM growth in vivo by inhibiting glioma cell proliferation
and promoting apoptosis. In addition, the Examples show for the
first time that depletion of CD44 or CD44-Fc fusion proteins
sensitizes GBM cells to chemotherapeutic and targeted agents in
vivo, rendering it an attractive therapeutic target for gliomas,
colon, breast, prostate, lung, melanoma, pancreatic cancer, liver
cancer, head and neck carcinoma, and ovarian cancers. CD44
antagonists, in the form of human soluble CD44-Fc fusion proteins,
such as hsCD44s-Fc, hsCD44v6-v10-Fc, hsCD44v8-v10-Fc, or
hsCD44v3-v10-FC, and CD44-specific shRNAs proved to be effective
therapeutic agents in inhibiting growth of human glioblastoma,
colon, breast, prostate, lung, melanoma, pancreatic cancer, liver
cancer, head and neck carcinoma, and ovarian cancers in mouse
models. shRNAs of CD44 can also be used as gene therapy and
delivered by nanoparticles.
[0335] The present Examples demonstrate for the first time that
CD44 functions upstream of mammalian Hippo stress and apoptotic
signaling pathway (merlin-MST1/2-Lats1/2-YAP-cIAP1/2) and of two
other downstream stress kinases, JNK and/or p38, along with their
effectors, p53, and caspases (FIG. 5 and FIG. 6). They also provide
evidence that CD44 plays an essential role in attenuating
activation of stress and apoptotic signaling pathways induced by
chemotherapeutic agents and reactive oxygen species (ROS) whereas
loss of CD44 function leads to their sustained activation that
promotes apoptosis of GBM cells and other cancer cells (see working
model in FIG. 10).
[0336] These Examples show that depletion of CD44 inhibits Erk1/2
activation induced by EGFR ligands and HGF but not by NGF or FBS
(FIG. 7), suggesting that CD44 serves as a co-receptor for these
RTKs and enhances their signaling activity in malignant glioma
cells and other cancer cells alike. Although the precise mechanism
whereby CD44 regulates RTK signaling requires further
investigation, its function as an HA receptor provides a possible
explanation. CD44 forms large aggregates on the cell surface upon
engagement by its multivalent ligand, HA. These aggregates often
reside in lipid rafts or other specialized membrane domains where
initiation of multiple signaling events occurs. In addition, CD44
can be expressed as a cell surface proteoglycan that binds numerous
heparin binding growth factors including HB-EGF and basic FGF. As
an RTK co-receptor, CD44 can therefore enhance signaling by
facilitating RTK oligomerization and presenting the appropriate
ligands to the corresponding RTKs. The ability of CD44v3-v1-Fc
fusion proteins to modulate bioactivity of heparin binding growth
factors, which include EGF family ligands, tumor angiogenic factors
such as VEGF, bFGF, and angiopoietins, suggests that CD44
antagonists can be used to successfully in many combination
therapies that target these ligands, their corresponding RTKs, and
their downstream signaling pathways.
[0337] CD44 antagonists, hsCD44-Fc fusion proteins, and in
particular hsCD44s-Fc, hsCD44v8-v10-Fc, or hsCD44v3-v10-Fc
constructs, displayed potent activity against GBM, breast cancer,
prostate cancer, melanoma, head-neck cancer, liver cancer, and
pancreatic cancer in mouse models and inhibited self-renewal of
breast and ovarian cancer stem cells, which offers hope for
eradicating these deadly cancers in the future. Human sCD44-Fc
fusion proteins may not only interfere with the function of CD44
expressed by GBM cells and other cancer cells but also with that
expressed by host cells infiltrated the tumors. These host cells
likely provide an essential contribution to progression of these
cancer types similar to types of the effects of other molecules on
cancers (Budhu et al., 2006; Orimo et al., 2005).
[0338] Currently available first line treatment options for human
GBM are chemo- and radiation therapy, although both are largely
palliative (Chamberlain, 2006). Effective treatments for malignant
melanoma, lung cancer, and pancreatic cancer are almost not
existed. There is also lack of effective treatment for liver
cancer, head-neck cancer, late stage colon cancer, late
stage/drug-resistant breast and prostate cancer. One hope for a
better clinical outcome is to identify targets that play essential
roles in mediating the microenvironment-derived survival signal and
drug-resistance and that their antagonists can sensitize responses
of these tumor cells to radiation, chemotherapeutic and targeted
drugs. The Examples show that CD44 plays an important role in
protecting cancer cells from oxidative and cytotoxic stress-induced
apoptotic signaling while enhancing RTK signaling suggesting that
CD44 may serve as an ideal therapeutic target to sensitize
malignant glioma and other types of cancer cells to radiation,
chemotherapy, and targeted therapies.
[0339] These Examples and Figures indicated that CD44 is a prime
target for a variety of human cancer types including but not
limited to human glioblastoma, colon cancer, breast cancer,
prostate cancer, lung cancer, melanoma, head-neck cancer, liver
cancer, pancreatic cancer, and ovarian cancer that CD44 antagonists
including CD44-Fc fusion proteins and shRNAs are potent anti-cancer
agents when used as single agents and in combinations with chemo-
and/or radiation therapy, and the targeted therapies against erbB
receptors, c-Met, IAPs, and activating p53. These Examples and
Figures also demonstrate that CD44-Fc fusion proteins sensitize
cancer cells to such cytotoxic agents such as chemo- and/or
radiation therapies. Therefore, these fusion proteins are
particularly amenable to being combined with such agents that will
induce and/or promote stresses in tumor cells.
[0340] These Examples and Figures also show that these CD44-Fc
fusion proteins bind specifically to HA and therefore can be used
to detect HA in tissues section and in body fluids (blood, plasma,
serum, and urine) for example in cancerous tissue. As a result,
these fusion proteins can be used to diagnose cancers in which HA
levels are elevated, which may lead to earlier detection of cancer
then currently available methods and save lives. These fusion
proteins can also be used to detect elevated HA levels, which will
valuable in prognosis and early assessments of efficacy of
therapeutic treatments, likely leading to more effective
personalized treatment plans that increase overall survival of
patients. The level of CD44 in these tumor samples and body fluid
samples can be assessed in conjunction with HA levels to achieve
more accurate predictions. Measuring HA and CD44 levels can be done
using standard immunological techniques and detection methods.
[0341] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0342] It is further to be understood that all values are
approximate, and are provided for description.
[0343] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
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